Research Article A 3D Porous Gelatin-Alginate-Based-IPN

Research ArticleA 3D Porous Gelatin-Alginate-Based-IPN Acts asan Efficient Promoter of Chondrogenesis from HumanAdipose-Derived Stem Cells

Sorina Dinescu1 Bianca Galateanu12 Eugen Radu3 Anca Hermenean2 Adriana Lungu4

Izabela Cristina Stancu4 Dana Jianu56 Tudorita Tumbar7 and Marieta Costache1

1Department of Biochemistry andMolecular BiologyUniversity of Bucharest 91-95 Splaiul Independentei 050095Bucharest Romania2Institute of Life Sciences Vasile Goldis Western University of Arad 86 Rebreanu 310414 Arad Romania3Molecular Biology and Pathology Research Lab ldquoMolimagexrdquo University Hospital Bucharest 169 Splaiul Independentei050098 Bucharest Romania4Advanced Polymer Materials Group University Politehnica of Bucharest 1-7 Gheorghe Polizu Street District 1011061 Bucharest Romania5Proestetica Medical SRL 38-40 T Stefan Street 011658 Bucharest Romania6International Society of Regenerative Medicine and Surgery 38-40 T Stefan Street 011658 Bucharest Romania7Department of Molecular Biology and Genetics Cornell University Ithaca NY 14853 USA

Correspondence should be addressed to Marieta Costache marietacostacheyahoocom

Received 31 October 2014 Accepted 18 January 2015

Academic Editor Junjun Fan

Copyright copy 2015 Sorina Dinescu et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cartilage has limited regeneration potential Thus there is an imperative need to develop new strategies for cartilage tissueengineering (CTE) amenable for clinical use RecentCTE approaches rely on optimal cell-scaffold interactions which require a greatdeal of optimization In this studywe attempt to build a novel gelatin- (G-) alginate- (A-) polyacrylamide (PAA) 3D interpenetratingnetwork (IPN) with superior performance in promoting chondrogenesis from human adipose-derived stem cells (hADSCs) Weshow that our G-A-PAA scaffold is capable of supporting hADSCs proliferation and survival with no apparent cytotoxic effectMoreover we find that after exposure to prochondrogenic conditions a key transcription factor known to induce chondrogenesisnamely Sox9 is highly expressed in our hADSCsG-A-PAAbioconstruct alongwith cartilage specificmarkers such as collagen typeII CEP68 and COMP extracellular matrix (ECM) components These data suggest that our G-A-PAA structural properties andformulation might enable hADSCs conversion towards functional chondrocytes We conclude that our novel G-A-PAA biomatrixis a good candidate for prospective in vivo CTE applications

1 Introduction

Articular cartilage has a very limited capacity for intrinsichealing Because the tissue is avascular the injury does notinduce the typical wound healing response characterized byinflammation and invasion of undifferentiated mesenchymalcells [1] Depending on the nature and severity of the cartilagelesion therapies currently used in cartilage tissue engineering(CTE) can either be directed towards tissue repair andreconstruction or towards local tissue regeneration [2] To

date strategies elaborated to solve cartilage defects remainineffective

The underlying principle of cell-based cartilage regener-ation therapy is based on the ability of stem cells or cartilageprecursor cells directly injected to the injury site to convertinto functional chondrocytes able to synthesize cartilagi-nous extracellular matrix (ECM) Cell-based regenerativetherapies appear to be more efficient than classical surgicalmethods rapidly providing the patient with better quality oflife However in the long term these regenerative strategies

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 252909 17 pageshttpdxdoiorg1011552015252909

2 Stem Cells International

did not prove superior to surgical proceduresThis highlightsthe crucial need to optimize the cell type appropriate scaffoldprochondrogenic factors added in the microenvironmentand the interaction between these components

One of the most difficult tasks in current cell-basedcartilage regenerative therapies is to identify the most suit-able matrix corresponding to each cartilage tissue defectTo develop stable and efficient strategies for CTE severalmaterial design approaches have been investigated [3ndash5]The substrate scaffold should correspond to certain criteriain order to be appropriate for CTE (i) appropriate 3Dstructure in order to recreate the in vivo microenvironment(ii) appropriate porosity to allow cell migration and diffusionof molecules nutrients and oxygen (iii) biocompatibility(iv) biodegradability (v) matrix design to allow its insertionby mini-invasive procedures [6]

Cell types found to be useful in cartilage repair strategiesare represented either by differentiated (chondrocytes) orundifferentiated cells (embryonic stem cells mesenchymalstem cells adipose-derived stem cells [7] synovium-derivedstem cells and periosteum-derived progenitor cells) Iden-tification of the correct cell source is a key aspect for acell-based cartilage reconstruction strategy since these cellsmust exhibit high chondrogenic potential From this pointof view each cell type from stem cells to chondrocytespresents certain advantages and disadvantages leaving themost efficient cell source still debatable

Mature chondrocytes represent possible candidates forcartilage repair due to their ability to produce type II collagensulfated glycosaminoglycans (GAG) and other moleculesspecific for the cartilaginous ECM [8] In the articular carti-lage only 1ndash5 of the volume is occupied by chondrocyteswhich are sparsely spread within the self-secreted ECMmade of collagens proteoglycans and other noncollagenousproteins [9] Considering the above mentioned cell-ECMratio although each chondrocyte has a high activity [10]the overall metabolic rate of the cartilage tissue is low andconsequently conducts to a poor self-regeneration potential

In contrast to primary chondrocytes mesenchymal stemcells (MSCs) are more abundant within certain tissuesmaking themeasier to isolateMain sources forMSC isolationare bone marrow (BM-MSCs) and adipose tissue but MSC-like cells were also identified in other tissue types includingsynovium periosteum skeletal muscle umbilical cord veinor placenta [11] These cells are all capable of chondro-genic differentiation in appropriate culture environments[12] Human adipose-derived stem cells (hADSCs) couldrepresent a viable source of mesenchymal-like stem cells forCTE applications due to their easy harvest [13 14] highchondrogenic potential [13 15 16] and wound healing prop-erties [17ndash20] Despite significant progress obtaining fromhADSCs functional chondrocytes capable of secreting type IIcollagen in tridimensional scaffolds remains challenging [21]

Current regenerative strategies in CTE aim to developappropriate scaffold capable of supporting chondrogenesisThe aim of this study is to evaluate the in vitro biological per-formance of anovel 3D porous scaffold in terms of biocom-patibility and the ability to support hADSCs chondrogenic

differentiation Specifically we developed a tricomponentsystem based on interpenetrating polymer networks (IPNs)of gelatin (G) alginate (A) and polyacrylamide (PAA) Westudied the evolution of prochondrogenic induced hADSCscommitment towards functional chondrocytes inside this 3Dporous scaffold which proved to be biocompatible and topossess physical and chemical properties similar to cartilageThis work contributes to current efforts to address thechallenges of using smart biomatrices to unleash the potentialof stem cells for curative CTE

2 Materials and Methods

21 Scaffold Synthesis Gelatin B (G) from bovine skin(Sigma) was used as 20 (wv) aqueous solution Sodiumalginate (SA) was used as 4 (wv) aqueous solution Acryl-amide (AA) for electrophoresis gt99 (HPLC) and NN1015840-methylenebis(acrylamide) (MBA) 99 triethanolamine(TEA) ammonium persulphate (APS) glutaric aldehyde(GA) as aqueous solution 25 and calcium chloride anhy-drous (CaCl

2) were purchased from Sigma and used without

further purificationIPNs based on G calcium alginate (A) and polyacry-

lamide (PAA) were prepared using a three-step procedurereported elsewhere [22] The three components were used ina weight ratio of 14 1 20 between G SA and AA with atotal solid content (T) of 21 Initially cross linked PAA(molar ratio between MBA and AA was 18 100) was gen-erated through network-forming radical polymerization ofsynthetic monomers in the presence of the natural polymersRedox initiation based on APS (1 molar with respect toAA and MBA) and TEA (12 molar with respect to APS)was used to perform the polymerization reaction undermild conditions Briefly the polymerization stock solutionwas prepared based on a protocol detailed elsewhere [22]through the dissolution of AA MBA and the correspondingamount of initiator in distilled water under stirring at roomtemperature (RT) 1mL of this solution was further mixedwith 8mL of gel solution and with 1mL of SA solution at40∘C The resulting viscous solution was degassed using anultrasound bath (Elma S 30H Elmasonic) for 15 minutes at40∘CThe polymerization accelerator TEA was added undervigorous stirring The network-forming polymerization ofAA and MBA was allowed to occur in Petri dishes for 24hours at 40∘C Semi-IPNs consisting in cross-linked PAAand uncross-linked G and SA were obtained Then thematerials were cooled for 2 hours at 4∘C to allow physicalgelation of G G was cross-linked through immersion in GA05 for 24 hours at RT followed by the cross-linking ofSA in a 1 CaCl

2aqueous solution for 24 hours Calcium

alginate (A) is thus formedThe resulting G-A-PAA hydrogelwas further purified in distilled water at 40∘C for four daysGravimetric measurement was used to assess the successof the IPNs formation through gel fraction (GF) analysisThe experiment consists in the assessment of GF as rationbetween remaining gel (dried) after extensive extraction ofthe eventual soluble fraction in distilled water at 40∘C with

Stem Cells International 3

respect to the total mass of obtained G-A-PAA product(dried) following the synthesis

GF =119898gel

1198980

times 100 (1)

Freeze-drying is becoming a common procedure to gen-erate porous scaffolds from highly hydrated materials andtherefore we used it to obtain porous G-A-PAA [23]

Control G-A and PAA hydrogels with the same T(21) were synthesized using similar procedures GF valueswere estimated also for these samples Porous scaffolds weregenerated through freeze-drying

22Morphological Assessment of Porosity through SEM Mor-phological characterization of the scaffolds was obtainedthrough Scanning ElectronMicroscopy (SEM) analysis of thegold-coated cross sections of the freeze-dried hydrogels Thestudy was performed using a QUANTA INSPECT F SEMdevice equipped with a field emission gun (FEG) with 12 nmresolution and with an X-ray energy dispersive spectrometer(EDS) Porous hydrogels were imaged prior to and aftercell-seeding (7 days after seeding) The samples were fixedusing 4 p-formaldehyde and subjected to freeze-dryingprocedure

23 Cell CultureModel Thehuman subcutaneous abdominalwhite adipose tissue was obtained from overweight womenundergoing elective liposuction All the medical procedureswere performed in compliance with theHelsinki Declarationwith the approval of Proestetica Medical Center Committee(reference number 11223102013) All subjects were in goodhealth and provided their written consent before participat-ing in the study The lipoaspirates (LAs) were immediatelyprocessed by collagenase digestion and the obtained suspen-sion was centrifuged at 2400 rpm for 10min Then the pelletwas resuspended in Dulbeccorsquos modified Eaglersquos medium(DMEM Sigma-Aldrich Co) supplemented with 40 fetalbovine serum (FBS) During 24 h the adherent stem cells wereallowed to attach to the culture surface and then the culturemedium was changed to DMEM containing 10 FBS Afterreaching 3rd passage of culture the specific mesenchymalstem cells surface markers panel CD29+ CD44+ CD73+CD90+ CD105+ CD14minus CD31minus and CD45minus was confirmedby flow cytometry

24 Cell-Scaffold Bioconstruct Achievement hADSCs in the3rd passage were seeded on top of G-A G-A-PAA and PAAbiomatrices at an initial density of 25 times 105 cellscm2 for bio-compatibility studies and of 106 cellscm2 for chondrogenicdifferentiation assessment The cell suspension was allowedto diffuse through the IPNs for 1 hour in order for the cellsto adhere to the inner layers of the biomaterial The porous3D biohybrids resulted after the scaffolds were put in contactwith hADSCs These are further referred to as hADSCsG-AhADSCsG-A-PAA and hADSCsPAA bioconstructs

For biocompatibility assessment the biohybrids wereincubated in standard conditions of cultivation inMesenPRORS Medium for 7 days with the medium refreshed every 2

days Regarding the chondrogenic differentiation protocol24 hours after seeding the bioconstructs were exposed toprochondrogenic conditions for 28 days using StemProChondrogenesis Differentiation Kit (Gibco Life Technolo-gies Foster City CA) hADSCs potential of differentiationwas assessed at 3 7 14 and 28 days after induction Thetime point when the systems were first exposed to thechondrogenic cocktail was considered T0

25 Spectrophotometric Assays

251 MTT Assay hADSCs capacity to proliferate into G-A G-A-PAA and PAA biomatrices was quantitatively deter-mined using MTT spectrophotometric assay at 24 h and 7days after seeding In this context all cell-scaffold biocon-structs were incubated in 1mgmL MTT (Thiazolyl BlueTetrazolium Bromide) solution (Sigma Aldrich Co Stein-heim Germany) for 8 hours The resulted formazan crystalswere observed in contrast phase using a Nikon Eclipse TS100 microscope (Nikon Instruments Europe AmsterdamNetherlands) and then subjected to solubilisation in iso-propanol for 2 hoursThe absorbance of the resulting solutionwas measured by spectrophotometry at 550 nm (AppliskanThermo ScientificWalthamMAUSA)The optical densitiesobtained are proportional to cell viability

252 LDH Assay The LDH assay is based on the quantifica-tion of the cytosolic lactate dehydrogenase enzyme releasedin the culture medium by the cells with damaged membrane

The environmental cytotoxic potential of the G-A G-A-PAA and PAA materials on the hADSCs was evaluatedusing ldquoIn vitro toxicology assay kit lactate dehydrogenasebasedrdquo (Sigma Aldrich Co Steinheim Germany) accordingto the manufacturerrsquos protocol Briefly the culture mediawere harvested at 24 h and 7 days after seeding and theywere mixed with the solutions provided in the kit After 20minutes of incubation at room temperature and darknessthe reaction was stopped with 1N hydrochloric acid (HCl)The LDH concentration was determined by measuring theoptic density of the resulting solutions at 490 nm (AppliskanThermo Scientific Waltham MA USA)

253 Quantitative ELISA Cartilage oligomeric matrix pro-tein (COMP) protein expression was assessed using Quan-tikine ELISAHumanCOMP (RampD Systems Abingdon UK)following manufacturerrsquos instructions COMP levels weredetermined from culture supernatants collected at T0 3 7 14and 28 days of culture in chondrogenic conditions and storedat minus20∘C Final results of the immunoassay were determinedbymeasuring the samples optical density at 450 nm and usinga 540 nm wavelength correction Additionally a standardcurve has been generated using the standard provided in thekit and following the assay procedure instructions

26 Fluorescence Labeling Assays

261 LiveDead Assay LiveDead fluorescence microscopyassay was performed to evaluate hADSCs viability and


Table 1 Primer sequences used to identify early and late chondrogenic markers

Target Nucleotide sequence Fragment lengthSox9 F 51015840-TTGAGCCTTAAAACGGTGCT-31015840 244 bpSox9 R 51015840-CTGGTGTTCTGAGAGGCACA-31015840

Col2a1 F 51015840-TCACGTACACTGCCCTGAAG-31015840 213 bpCol2a1R 51015840-TGCAACGGATTGTGTTGTTT-31015840

CEP68 F 51015840-TCTTGTCCCATGGAGAGTCC-31015840 154 bpCEP68 R 51015840-GCCCCACTCTTCTTGGTGTA-31015840

COMP F 51015840-CCATGGACTTGGCCAGTAGG-31015840 385 bpCOMP R 51015840-GAGGAAGAGCCAGTGACGAAA-31015840

TBP F 51015840-AGGCATCTGTCTTTGCACAC-31015840 166 bpTBP R 51015840-GGGTCAGTCCAGTGCCATAA-31015840

YWHAZ F 51015840-AGGAGATAAAAAGAACATCCAGTCA-31015840 269 bpYWHAZ R 51015840-TATTCTCGAGCCATCTGCTGTTT-31015840

proliferation within the 3D culture systems using theLiveDead Kit (Invitrogen Life Technologies Foster CityCA) This fluorescence-based kit combines calcein AM andethidium bromide to yield two-color discrimination of thepopulation of live cells from the dead-cell population

Briefly at 24 h and 7 days after seeding hADSCsG-AhADSCsG-A-PAA and hADSCsPAA bioconstructs wereincubated with a staining solution prepared according tomanufacturerrsquos instructions for 15 minutes in the darkNext the stained 3D cultures were analyzed by fluorescencemicroscopy using an Olympus IX71 inverted microscope andimages were captured with Cell F Imaging Software (Olym-pus Hamburg Germany 2008) 3D images were capturedusing confocal microscopy (Leica TCS-SP5 confocal scannersystem)

262 Immunofluorescence and Actin Filaments Fluores-cent Labeling The protein expression of actin and tubulincytoskeleton proteins and of the nuclear transcription factorSRY (sex determining region Y) box 9 (Sox9) collagentype II and the centrosomal protein of 68 kDa (CEP68)chondrogenic specific markers were studied by confocal flu-orescence microscopy using a Carl Zeiss LSM710 laser scan-ning microscope with Zeiss 20x and 40x 05NA objectiveshADSCs morphology and cytoskeleton fibers distribution incontact with G-A and G-A-PAA scaffolds were studied at48 h days after seeding while chondrogenic specific markerswere assessed at T0 3 7 14 and 28 days post-hADSCsG-A and hADSCsG-A-PAA exposure to the prochondrogenicenvironment

In order to fluorescently label the target proteins bothhADSCsG-A and hADSCsG-A-PAA constructs were fixedwith 4 PFA for 8 h and permeabilized with 2 BSA01Triton X-100 solution at 4∘C Next the constructs wereincubated 4 h at 37∘C with Alexa Fluor 488 Phalloidin(Molecular Probes Life Technologies Foster City CA) foractin labeling and overnight at 4∘C with mouse clonal anti-Sox9 (Abcam Cambridge UK) goat polyclonal anti-CEP68and goat polyclonal anti-Col2a1 (Santa-Cruz BiotechnologyHeidelberg Germany) antibodies The bioconstructs were

further incubated in TRITC conjugated goat anti-mouseand FITC conjugated rabbit anti-goat secondary antibodiessolutions for 1 h (Santa-Cruz Biotechnology HeidelbergGermany) After cell nuclei were stained with DAPI for30min the resulting labeled constructs were inspected inconfocal fluorescence microscopy Carl Zeiss Zen 2010 soft-ware version 60 was used for image acquisition and analysisThe 405 488 and 543 nm laser lines were used for excitationand fluorescence emission was detected at 490ndash515 nm forDAPI 520ndash550 nm for FITC and 600ndash680 nm for TRITCThe confocal aperture used corresponded to a back-projectedsize of 1 Airy unit Some of the images were acquired as z-stacks and a maximal projection algorithm was used for 3Dreconstruction

27 qPCR Quantification hADSCsG-A and hADSCsG-A-PAA bioconstructs were cut into fragments and the totalRNA was isolated using TRIzol Reagent (Invitrogen Fos-ter City CA USA) in accordance with the manufacturerrsquosinstructions After the isolated total RNA was tested forpurity and concentration on NanoDrop spectrophotometer(Shimadzu Duisburg Germany) and for integrity on theBioAnalyzer 2100 (Agilent Technologies Waldbronn Ger-many) one microgram of total cellular RNA was reverse-transcribed to corresponding cDNA using iScript cDNASynthesis kit (BioRad Hercules CA USA) in a reactionvolume of 20 120583L Real-Time RT-PCR was performed on aLightCycler 20 carrousel-based system using LightCyclerFast Start DNAMaster SYBRGreen I Kit (RocheMannheimGermany) Thermal cycling conditions were 10min at 95∘Cfollowed by 45 cycles of 10 s at 95∘C 10 s at 56∘C and 20 s at72∘C The sequences of the primers used for Sox9 collagentype II CEP68 and COMP genes detection are presentedin Table 1 and were purchased from Life Technologies ThemRNA expression levels of target genes were normalizedto the levels of TATA binding protein (TBP) and Tyrosine3-monooxygenasetryptophan 5-monooxygenase activationprotein (YWHAZ) which were used as reference genes andwere assessed in the same experimental conditions Addition-ally mRNA levels of the target chondrogenic markers were


(a) (b)

(c) (d)

Figure 1 SEMmicrographs showing the porosity of the unseeded scaffolds as visible through cross sections (a) G-A-PAA (b) G-A (c) PAAand (d) surface of PAA negative control

compared to the mRNA levels of the same markers from aculture ofmature human chondrocytes as a reference sample

28 Statistical Analysis The spectrophotometric and geneexpression data were statistically analyzed using GraphPadPrism 303 Software one-way ANOVA and Bonferroni testwhile comparison between chondrogenic markers mRNAlevels of expression was performed using GenEx Enterprise542 software The experiments were performed with 119899 = 3biological replicates and each data set is presented as theaverage of three replicates (mean plusmn standard deviation)

3 Results

31 IPN Formation To evaluate the success of the IPNsformation GF values were calculated based on gravimetricmeasurements and using (1) The results indicated high GFvalues ranging from 95 plusmn 6 for G-A to 98 plusmn 3 for PAA and98 plusmn 2 for G-A-PAA hydrogels This confirms the successof the network formation through the described synthesisprocedure

32 Capacity to Generate Porous Structures Freeze-drying ofthe synthetized hydrogels generated porous materials withfoam-like appearance Morphological information on thepore shapes homogeneity sizes and interconnection is givenin Figure 1 as obtained from SEM investigation of the crosssections

G-A IPN (Figure 1(b)) presents numerous interconnectedpores with irregular still ovoidal shape and dimensionsof approximately 100 120583m times 40 120583m PAA (Figure 1(c)) alsopresents irregular pores with a different morphology withthicker separation walls and larger dimensional distributionExtremely interesting is that the studied G-A-PAA IPN(Figure 1(a)) does not combine the morphologies of the twocontrol samples but has a totally novel architecture withlarger pores (approximately 200120583m times 100 120583m) intercon-nected and with extremely thin and smooth separation walls

33 Biocompatibility Assessment of Novel Porous Biomatricesin Contact with hADSCs The biocompatibility of G-A-PAAnovel IPN was tested considering G-A as reference scaffoldand PAA as negative control respectively In this context weevaluated the matricesrsquo potential to support cellular viability


(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)

Figure 2 Confocal fluorescence microscopy (a) and SEM micrographs (b) showing G-A-PAA scaffold morphology 1 unseeded 2 at 24 hafter seeding 3 at 7 days after seeding G-A scaffold morphology 4 unseeded 5 at 24 h after seeding 6 at 7 days after seeding Confocalmicroscopy images show the autofluorescence of the matrix (green) and DAPI-stained nuclei (blue)

and proliferation by LiveDead and MTT assays as well astheir eventual cytotoxic effect using LDH test AdditionallyhADSCs distribution morphology and cytoskeleton orga-nization inside the porous scaffolds were highlighted usingSEM and confocal fluorescence microscopy

331 hADSCs Distribution inside the Porous Biomatri-ces SEM micrographs together with confocal fluorescence

microscopy were performed to confirm cell distributionshape and proliferation trend SEM analysis revealed thedistribution of the cells within the porous hydrogels and thematerials behavior in plain culture medium during one weekof incubation in standard conditions of culture Accordinglyas shown in Figure 2 after 7 days of culture the biomaterialsdisplayed an interconnected porous pattern and hADSCspopulated deep layers of the scaffolds Fluorescence confocal


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Confocal fluorescence microscopy micrographs of hADSCs actin filaments and tubulin network in hADSCsG-A-PAA biohybrid((a) (c)) and in hADSCsG-A control ((b) (d)) Matrix autofluorescence is seen in purple DAPI-stained nuclei are blue

microscopy was performed at 2 and 7 days after seeding ofthe hADSCs in the G-A-PAA and G-A scaffolds after DAPIstaining of nuclei Accordingly hADSCs proved to populatethe pores of the scaffolds after 2 days of culture and continuedto expand

Furthermore at 24 h of culture hADSCs seeded in bothG-A-PAA and G-A scaffolds displayed a spherical-like mor-phology (Figure 2 (a2)(b2) and Figure 2 (a5)(b5)) whileafter one week of incubation they adopted a characteristicspindle-like shape (Figure 2 (a3)(b3) and Figure 2 (a6)(b6))suggesting their possible adhesion to the materials

332 hADSCs Morphology and Cytoskeleton Organizationinside G-A-PAA and G-A IPNs hADSCs morphology andthe ability to interact with the substrate material in termsof adhesion and cytoskeleton development were carefullyinvestigated once the hADSCsG-A-PAA and hADSCsG-Abiohybrids were obtained 48 h after seeding hADSCs fromboth constructs displayed long and distinctive actin filamentssurrounding the nuclei which clearly determined cell overall

morphology (Figures 3(a) and 3(b)) Consequently hADSCsability to dynamically form microfilaments may be consid-ered part of the physiological shape modeling process inresponse to the stimulus represented by the substrate Theactin cytoskeleton underlies the cell adhesion process whichis highly important for further tissue formation

The actin cytoskeleton is closely related to the micro-tubule network regulating cell motility and maintenanceof cell shape Tubulin distribution (Figures 3(c) and 3(d))inside the hADSCs seededgrowncultured in the scaffoldsalso revealed their spindle-like shape and attachment to thesubstrate

333 LiveDead Fluorescence Microscopy Assay In orderto examine cell survival inside the tested biomaterials theviability of hADSCs was evaluated at 24 h and 7 days afterseeding by fluorescence (Figure 4) and confocal fluorescence(Figure 5) microscopy based on the simultaneous staining oflive (green labeled) and dead (red labeled) cells


(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m

Figure 4 Fluorescence microscopy micrographs revealing live and dead cells in G-A-PAA (a) G-A (b) and PAA (c) matrices at 24 h (1)5 days (2) and 7 days (3) of culture

xy z

8188 120583m000 120583m

Width 127279120583m height 127279120583m depth 8188 120583m

(a)

10808 120583m

000 120583m

xy z


(b)

Figure 5 3D laser scanning reconstruction of LiveDead stained (a) hADSCsG-A-PAA bioconstruct and (b) hADSCsG-A bioconstructViable (green) and dead (red) cells are seen embedded in the autofluorescent matrix (orange)

At 24 h after seeding bright green labeled cells wereobserved surrounding the G-A-PAA and G-A pores whileunattached spherical-shaped cells were displayed on top ofPAA matrix After one week of culture the number of green-labeled hADSCs inside both the sample and the controlscaffold increased as compared to 24 h after seeding confirm-ing their proliferation This observation could indicate that

G-A-PAA andG-A sustain cellular viability and proliferationoffering a proper microenvironment to hADSCs Regardingthe negative control the amount of cells seeded on top of thescaffold dramatically decreased after 7 days of culture

Laser scanning of hADSCsG-A-PAA and hADSCsG-A bioconstructs confirmed previously presented SEM celldistribution data (Figure 2) and in addition showed that


00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay

hADSCsG-A-PAAhADSCsG-A

hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)

Figure 6 The quantification of (a) hADSCs proliferation rate in G-A-PAA G-A and PAA biomatrices as revealed by MTT test and (b) thecytotoxic potential of G-A-PAA G-A and PAA biomatrices on hADSCs as revealed by LDH assay at 24 h 5 days and 7 days (lowast119875 lt 005(hADSCsG-A-PAA versus hADSCsG-A bioconstruct) lowastlowastlowast119875 lt 0001 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct) 119875 lt 0001(hADSCsG-A-PAA 5 days versus 24 h days and 7 days versus 5 days) 119875 lt 001 (hADSCsG-A 7 days versus 5 days) 119875 lt 0001(hADSCsG-A 5 days versus 24 h))

hADSCs inside pores were viable after one week of culture(Figure 5)

Furthermore we observed an increased number of viablegreen labeled hADSCs inside G-A-PAA scaffold as comparedto G-A control material which populated in large groups thepores of the matrix

334 MTT To confirm hADSCs viability and the prolifer-ation rate observed by fluorescence microscopy MTT assaywas employed as a more accurate approach In this contextthe hADSCsG-A-PAA hADSCsG-A and hADSCsPAAbioconstructs were subjected to MTT spectrophotometricassay at 24 h and 5 and 7 days of culture (Figure 6(a))

Our results showed that in PAA scaffold the level ofthe formazan concentration was almost undetectable duringthe entire experimental period This suggested that hADSCsdid not survive 24 h in contact with the negative controlIn contrast hADSCs inside G-A-PAA and G-A scaffoldsproliferated during one week of experiment with the samestatistical significance increase (119875 lt 0001) between 24 h and5 days after seeding During the 5th and the 7th day of culturethe number of the metabolically active cells inside G-A-PAAmatrix significantly increased (119875 lt 0001) when compared tohADSCs seeded in G-A control scaffold (119875 lt 001)

No significant differenceswere observed in cellular viabil-ity between hADSCsG-A-PAA and hADSCsG-A scaffoldsas indicated by their absorbance values measured at 24 hAt 5 days of culture a slightly significant increase of cellularviability (119875 lt 005) was detected for cells in G-A-PAAmatrixas compared to the control biohybrid while at 7 days afterseeding the viability difference between these bioconstructswas found significantly higher (119875 lt 0001)

335 LDH The cytotoxic potential of G-A-PAA scaf-fold was evaluated by spectrophotometric quantification of

the LDH enzyme release in the culture media by the embed-ded hADSCs G-A and PAA matrices preseeded with thesamenumber of hADSCswere used as reference andnegativecontrol respectively (Figure 6(b))

At 24 h after seeding LDH levels detected in the culturemedium harvested from the negative control biohybrid werefound statistically significantly higher (119875 lt 0001) ascompared to the results obtained for hADSCsG-A-PAA andhADSCsG-A biohybrids This observation together with thedetection of very low LDH levels in hADSCsPAA at 5 and 7days of culture conducted to the presumption that hADSCsdid not survive in contact with PAA scaffold more than 24 h

Although G-A-PAA scaffold displayed significantlyhigher (119875 lt 005) levels of LDH as compared to the controlbiomatrix at 24 h no differences were registered at 5 and7 days of culture between these samples Considering thisprofile the higher levels of LDH released in the culturemedium by hADSCs seeded in G-A-PAA at the beginning ofthe experiment could be determined by the unpolymerisedAA residues which were subsequently washed during thesubcultivation procedures

LDH concentration in hADSCsG-A-PAA and hADSCsG-A biohybrids was significantly higher at 5 days of culture(119875 lt 0001) than at 24 h in accordance to cell proliferationrates Consequently the overall ratio between proliferationand cellular death is maintained throughout the experimentAt 7 days of culture no significant differences were noticed ascompared to the previous experimental time point

34 hADSCs In Vitro Chondrogenic Differentiation Assess-ment inside G-A-PAA Scaffold The chondrogenic processin hADSCsG-A-PAA and hADSCsG-A bioconstructs wasmonitored for 28 days by the evaluation of early and latechondrogenic markers gene and protein levels of expression


341 qPCR Quantification of Sox9 CEP68 and COMPChondrogenic Markers Molecular analysis of Sox9 collagentype II (Col2a1) CEP68 and COMP chondrogenic markersgene expression at 3 7 14 and 28 days after induction revealeda complex transcriptional and signaling pathway underlyingthe chondrogenic differentiation process (Figure 7) A sampleharvested before the bioconstructs exposure to chondrogenicconditions was used as reference for gene expression analysisand will be further addressed as T0

Sox9 was shown to play an essential role in cell differ-entiation to the chondrogenic pathway and in chondrocytefate thus being considered the master regulator and keyinducer of the chondrogenic differentiation process In ourexperiment Sox9 mRNA high levels were detected at 3 daysafter induction in both hADSCsG-A-PAA and hADSCsG-A bioconstructs suggesting its activation as compared tothe basal levels registered at T0 (Figure 7(a)) Interestinglya statistically significantly higher level of Sox9 transcript(119875 lt 005) was found in hADSCsG-A-PAA than in thecontrol biohybrid after 3 days of experiment Furthermorea significant upregulated Sox9 expression was found bothin hADSCsG-A-PAA bioconstruct (119875 lt 0001) and incontrol (119875 lt 0001) at 7 days postchondrogenic inductionas compared to the levels detected at 3 days Together withthis ascending expression profile found for the first weekof experiment corresponding to the initial activation ofSox9 chondrogenic inducer Sox9 mRNA levels at 7 dayswere also found to be statistically significantly higher (119875 lt001) in hADSCsG-A-PAA than in the control bioconstructAdditionally this difference was also present at 14 days ofinduced chondrogenesis since Sox9 transcript levels wereproved to remain constant between 7 and 14 days of experi-mentduring the secondweek of experiment Notably 28 dayspostchondrogenic induction Sox9 mRNA levels registered asignificant decrease (119875 lt 0001) in both biohybrids while noimportant difference was found between hADSCsG-A-PAAand hADSCsG-A in terms of Sox9 pattern of expression

In our particular conditions Col2a1 gene expressionwas first detected at 3 days of induced chondrogenesis inhADSCsG-A-PAA bioconstruct by comparison to Col2a1levels at T0 (119875 lt 005) suggesting that this ECM markersynthesis begins early during the chondrogenic differen-tiation process (Figure 7(b)) In contrast collagen type IIwas statistically significant expressed for the first time inthe control bioconstruct at 7 days after induction (119875 lt0001) Furthermore mRNA levels were found significantlyhigher in hADSCsG-A-PAA than in hADSCsG-A afterone week of in vitro chondrogenesis (119875 lt 001) Col2a1transcript levels statistically increased (119875 lt 0001) between7 and 14 days in both bioconstructs also maintaining thedifference in expression higher in hADSCsG-A-PAA thanin control (119875 lt 0001) This upregulated profile registereda constant and statistically significant increase (119875 lt 0001)in both bioconstructs during 14ndash28 days interval OverallCol2a1 gene expression profile registered an increasing trendsuggesting collagen type II continuous synthesis as proof ofECM accumulation

CEP68 was detected in samples harvested from both bio-constructs starting with day 7 of prochondrogenic induction

(Figure 7(c)) at statistically significantly higher levels (119875 lt005) in hADSCsG-A-PAA than in control AdditionallyCEP68mRNA levels of expressionwere found to be increasedat 7 days as compared to 3 days after induction bothin hADSCsG-A-PAA (119875 lt 0001) and in hADSCsG-A(119875 lt 001) systems The general gene expression profile ofCEP68 resembles Col2a1 trend registering a gradual increaseduring the 28 days of in vitro chondrogenesis Thus CEP68transcript levels significantly increased (119875 lt 0001) in bothbioconstructs between 7 and 14 days as well as in the lasttwo weeks of experiment while the statistically significantdifference (119875 lt 0001) in CEP68 gene expression betweenhADSCsG-A-PAA and hADSCsG-A constructs was alsomaintained constant

Similar to CEP68 ECMmarker COMP was first detected(119875 lt 0001) in hADSCsG-A-PAA system at 7 dayspostchondrogenic induction as compared to 3 days witha gene expression significantly higher (119875 lt 005) thanthe levels found in control (Figure 7(d)) COMP mRNAlevels gradually increased in both constructs between 7and 14 days (119875 lt 0001) of experiment maintaining astatistically significant difference (119875 lt 0001) betweenCOMPtranscript levels in hADSCsG-A-PAA and in hADSCsG-A Furthermore COMP expression considerably increased(119875 lt 0001) in hADSCsG-A bioconstruct after the secondweek whereas simultaneously the levels in hADSCsG-A-PAA registered a lower increase (119875 lt 001)

342 Qualitative Assessment of Sox9 Col2a1 and CEP68Chondrogenic Markers Sox9 Col2a1 and CEP68 proteinexpression was evaluated by confocal fluorescence micros-copy and the captured micrographs displaying the earliestpositive expression during the chondrogenic differentiationprocess are presented in Figure 8 A set of hADSCsG-A-PAAand hADSCsG-A bioconstructs unexposed to chondrogenicconditions were used as reference and proved the uncommit-ted state of the embedded hADSCs

As shown in Figure 8(a2) and Figure 8(b2) Sox9 tran-scriptional factor was first expressed at protein level at 3days postchondrogenic induction in both hADSCsG-A-PAAand hADSCsG-A bioconstructs However hADSCs in oursample were observed to condense in larger groups and toexpress more frequently Sox9 as compared to the cells inthe control matrix These observations are in accordancewith the data obtained after Sox9 gene expression analysis(Figure 7(a))

ECM synthesis is a specific process during chondrogen-esis The expression of Col2a1 and CEP68 ECM proteins islate chondrogenic marker suggesting stem cell conversiontowards mature chondrocytes In our experimental condi-tions both Col2a1 and CEP68 were first expressed after 7days of chondrogenic induction as also revealed by qPCRresults (Figures 7(b) and 7(c)) In addition using confocalfluorescence microscopy net differences in cellular distri-bution inside G-A-PAA and G-A matrices were highlightedafter one week of chondrogenesis hADSCs inside G-A-PAA


T0 3d 7d 14d 28d

hADSCsG-AhADSCsG-A-PAA

00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)

Figure 7 Gene expression profiles of (a) Sox9 (lowast119875 lt 005 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 3 days) lowastlowast119875 lt 001(hADSCsG-A-PAA versus hADSCsG-A bioconstruct 7 days and 14 days) ∙∙∙119875 lt 0001 (hADSCsG-A 7 days versus 3 days and 28 daysversus 14 days) 119875 lt 0001 (hADSCsG-A-PAA 7 days versus 3 days and 28 days versus 14 days)) (b) Col2a1 (lowastlowast119875 lt 001 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 7 days) lowastlowastlowast119875 lt 0001 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 14 days and 28 days)∙∙∙119875 lt 0001 (hADSCsG-A 7 days versus 3 days) 119875 lt 005 (hADSCsG-A-PAA 3 days versus T0) 119875 lt 0001 (hADSCsG-A-PAA 7 days

versus 3 days)) (c) CEP68 (lowast119875 lt 005 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 7 days) lowastlowastlowast119875 lt 0001 (hADSCsG-A-PAAversus hADSCsG-A bioconstruct 14 days and 28 days) ∙∙119875 lt 001 (hADSCsG-A 7 days versus 3 days) 119875 lt 0001 (hADSCsG-A-PAA 7days versus 3 days)) (d) COMP (lowast119875 lt 005 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 7 days) lowastlowast119875 lt 001 (hADSCsG-A-PAAversus hADSCsG-A bioconstruct 28 days) lowastlowastlowast119875 lt 0001 (hADSCsG-A-PAA versus hADSCsG-A bioconstruct 14 days) ∙∙∙119875 lt 0001(hADSCsG-A 28 days versus 14 days) 119875 lt 001 (hADSCsG-A-PAA 28 days versus 14 days)) as revealed by qPCR analysis and GraphPadPrism 30 data statistical analysis

scaffold were found to be organized in large clusters andtrapped in their self-secreted ECM whereas differentiatinghADSCs inside G-A control matrix barely assembled in smallgroups as shown by the double staining of cell nuclei andECM proteins (Figure 8(a3) (a4) and Figure 8(b3) (b4))

343 Quantitative Detection of COMP Protein ExpressionCOMP protein expression was quantitatively evaluated inboth bioconstructs using ELISA immunoassay The results(Figure 9) revealed an increasing trend of COMP proteinexpression between 7 and 28 days of induced chondrogenic


(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m

Figure 8 Confocal microscopy micrographs of cells inside (a)G-A-PAA and (b) G-A scaffolds during chondrogenesis (1) 24 hafter seeding (2) Sox9 positive expression (red fluorescence) at 7days postchondrogenic induction (3) Col2a1 positive expression(green fluorescence) at 14 days postchondrogenic induction and(4) CEP68 positive expression (green fluorescence) at 14 dayspostchondrogenic induction

differentiationThedifference (119875 lt 005) registered in proteinlevels at 7 days between hADSCsG-A-PAA and hADSCsG-A biohybrids increased to a higher significance (119875 lt 0001)during the last two weeks of experiment COMP expressionsignificantly increased (119875 lt 0001) between 7 and 14 daysin the control system and then registered a lower increase(119875 lt 001) for the last experimental interval whereas theincrease in COMP protein expression in hADSCsG-A-PAAbioconstruct was found to be statistically significant (119875 lt0001) and constant in both time intervals

The data obtained for COMP protein expression arein total accordance with COMP gene expression profileconfirming COMP relation to ECM accumulation and itsstatus as a late chondrogenic marker

0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙

Figure 9 COMPexpression in hADSCsG-A-PAAand hADSCsG-A bioconstructs exposed to chondrogenic conditions for 28 days asrevealed by ELISA immunoassay (lowast119875 lt 005 (hADSCsG-A-PAAversus hADSCsG-A 7 days) lowastlowastlowast119875 lt 0001 (hADSCsG-A-PAAversus hADSCsG-A 14 days and 28 days) ∙∙119875 lt 001 (hADSCsG-A 28 days versus 14 days) ∙∙∙119875 lt 0001 (hADSCsG-A 14 days versus7 days) 119875 lt 0001 (hADSCsG-A-PAA 14 days versus 7 days and28 days versus 14 days))

4 Discussion

Articular cartilage is a highly specialized tissue which hasa particular function in protecting the bone ends from theforces associated with high mechanical load thus reducingjoint friction Due to its aneural avascular and alymphaticstructure cartilage is a complex and particular type of tissue[24] which possesses a limited self-regeneration potentialDue to these cartilage characteristics strong clinical require-ments have conducted to the development of new strategiesfor cartilage tissue engineering (CTE)

In an effort to generate cartilage-like tissue a variety ofpolymers including collagen [25 26] gelatin [27] silk [28]alginate [29] hyaluronan [30] chitosan [31] agarose [32]polyethylene glycol [33] polyglycolide [34] poly(lactic-co-glycolic acid) and hybrids of synthesized or natural materials[35ndash37] have been tested to date Polymersrsquo major advantagesare the chemical physical and functional resemblance withmacromolecular constituents of the ECM Despite appropri-ate biodegradability and biocompatibility somemacromolec-ular components do not possess appropriate mechanicalproperties or biodegradation rate A combination of differentnatural and synthetic polymers could in principle provideappropriate biodegradability biocompatibility and surfacecharacteristics and mimic the appropriate microenviron-ment to support cell adhesion and chondrogenic differentia-tion [38 39] Particularly a tricomponent IPN based on G Aand PAA has been recently demonstrated to present tunableproperties with respect to water affinity biodegradabilitymechanical behavior and the capacity tomodulate the poros-ity [22] It has been shown that through the combination


of these three materials synergistic effects can be obtainedConsequently such IPNs are ideal for applications in soft TE

Previous reports have shown that both BM-MSCs andhADSCs provide attractive cell sources for CTE in vitro andin vivo [40] but hADSCs seem to possess more advantagesfor current clinical applications Several studies have shownthe potential use of hADSCs in different CTE approachesby demonstrating the chondrogenic differentiation potentialof these cells (i) when cultured using the typical aggregateculture technique (pellet culture) [41] (ii) when seeded inmaterials or (iii) when implanted in different animal models[42ndash44] Awad et al [45] compared several different scaffoldsbased on agarose alginate and gelatin as support materi-als for hADSC adhesion proliferation and chondrogenicdifferentiation and concluded that these cells were able todifferentiate into chondrocytes when cultured into any ofthese scaffolds [46]

In our studies we showed that G-A-PAA scaffold is agood candidate for cartilage tissue regeneration purposes dueto its good physical chemical and structural properties itsbiocompatibility and not least its prochondrogenic poten-tial With respect to the cell distribution shape and prolif-eration trend valuable information was obtained through acritical comparison of results obtained from complementarytechniques such as SEM and confocal microscopy outcomes(Figure 3) This corroboration also revealed the materialsrsquobehavior in plain culture medium during one week ofincubationWenoticed that the scaffoldsmaintained an inter-connected porous pattern after 7 days of incubation in plainculture medium (Figure 2(a1)(b1) and Figure 2(a4)(b4))This pattern allowed further hADSCs efficient penetrationin the deeper layers of the structures (Figure 2 (a3)(b3) andFigure 2 (a6)(b6)) On the other hand although negativecontrol PAA scaffoldrsquos inner structure was proved to beporous and highly structured (Figure 1(c)) a compact thinpolymer layer was noticed on the surface of the hydrogel(Figure 1(d)) This superficial film did not allow cellularinfiltration

Moreover the tricomponent G-A-PAA scaffold preservedthe highly ordered porosity pattern and the well-definedinterconnected pores when compared to the bicomponentG-A control matrix (Figure 2) probably due to the presenceof PAA in its formulation In addition the quantitativeand qualitative biocompatibility investigations performed onboth bioconstructs showed that our G-A-PAA engineeredsystem was able to support hADSCs survival adhesionand proliferation despite its synthetic PAA component Weconcluded that the presence of this polymer offered betterproperties toG-A-PAA scaffold in terms of structure stabilitypore dimensions and cell distribution as compared to thereference matrix

The actin and tubulin filaments revealed by confo-cal microscopy (Figure 3) confirmed hADSCs characteristicspindle-like morphology in contact with both biomaterialssuggesting cells capacity to adapt to the provided condi-tions Even with the presence of PAA synthetic compoundin its formulation G-A-PAA biomatrix displayed a propermicroenvironment that allowed hADSCs attachment andcytoskeleton dynamics Furthermore 3D reconstructions of

laser scanned hADSCsG-A-PAA and hADSCsG-A biocon-structs revealed a net positive ratio between living and deadcells inside the biomaterials

Interestingly grouped cell proliferation was observedin hADSCsG-A-PAA system during one week of cul-ture (Figure 2 (a3)(b3)) probably favored by the three-dimensional porous architecture of the scaffold Very impor-tantly at 24 h after seeding hADSCs followed the irregularshape ofG-A controlmatrix (Figure 2 (a5)(b5)) UnlikeG-A-PAA the bicomponent substrate only promoted proliferationwith the formation of a monolayer-like distribution (Figure 2(a6)(b6)) Considering that these scaffolds were designed forCTE applications and that cell condensation is a critical stepin stem cells commitment to chondrogenesis we concludedthat G-A-PAA IPN displays structural advantages over G-A control Consequently G-A-PAA biomatrix was used ashADSCs temporary artificial microenvironment for furtherin vitro chondrogenesis studies

Chondrogenesis is a multistep pathway during whichmesenchymal cells first commit to the chondrogenic cell fatesubsequently condense undergo morphological changesand turn on cartilage specific genes [47] Precursor cellscondensation is one of the early events in chondrogenesisThe commitment towards the chondrogenic lineage dependson some initiation signals represented by the cell-cell andcell-matrix interactions and it is associated with changes inthe cytoskeleton architecture and an increase in cell adhesion[48] Sox9 is one of the chondrogenic markers expressedby cells in early stages of condensation Sox transcriptionfactors were originally identified as an sry gene located onchromosome Y Sox factors contain a high-mobility-groupbox (HMG) domain which contributes to its attachment tothe small groove of DNA and to the interaction with othertranscription factors [49 50] Lefebvre et al [51] and Bell et al[52] previously demonstrated that Sox9 is a potent activatorof Col2a1 gene expression as it binds to a high-mobilitygroup (HMG) box present in the enhancer of Col2a1 genethus strongly promoting transcription of Col2a1 reportergenes The transcriptional complex includes Sox5 and Sox6which are also essential for chondrogenic development butcannot activate Col2a1 gene expression in the absence ofSox9 [53] Sox9 is essential for cell commitment towardschondrogenic pathway and for the activation of genes encod-ing chondrocyte-specific matrix proteins such as collagentype II (Col2a1) [54] aggrecan [55 56] matrilin-1 [57] andCOMP [58] These proteins are required for maintaining thebiochemical properties of articular cartilage

CEP68 is considered a new marker for the chondro-genic differentiation process which efficiently complementscollagen type II expression in regenerative CTE approaches[59 60] CEP68 functions as an ECM protein which allowsdiscrimination of chondrocytes from osteoblasts and MSCsin cell cultures [60]

COMP which is predominantly found in the ECM ofcartilage tendons and ligaments [61] is a member of theThrombospondin (TSP) calcium-binding protein family Itplays a crucial role in endochondral ossification and inthe assembly and stabilization of the ECMwhilemaintainingthe structural integrity of the cartilage through its interaction


1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11

28d hADSCsG-A-PAA28d hADSCsG-A14d hADSCsG-A-PAA14d hADSCsG-A7d hADSCsG-A-PAA

7d hADSCsG-A3d hADSCsG-A-PAA3d hADSCsG-AT0 hADSCsG-A-PAAT0 hADSCsG-A

(b)

Figure 10 Sox9 Col2a1 CEP68 and COMP chondrogenic markers gene expression analysis via GenEx software generated (a) a heat mapand (b) a scatter plot revealing 3D distribution of samples depending on the expression of related ECMmarkers Col2a1 CEP68 and COMP

with aggrecan fibronectin and matrilin [62ndash64] COMP isable to bind to collagens types I and II via its C-terminalglobular domain and thus it acts as a catalyst to promotefibril formation [65] Due to its interaction with cell adhesionmolecules COMP was found to favor cellular attachment[66] and also to initiate the transition from chondroprogen-itor cells to fully committed chondrocytes thus enhancingchondrogenesis [48]

In our in vitro approach hADSCs underwent chondro-genic induction by growth factors in both G-A and G-A-PAAscaffolds demonstrating upregulation of cartilage specificgenes and the synthesis of cartilaginous proteins In ourexperiment Sox9 was investigated as an early chondrogenicmarker and was found to be expressed at higher levels inhADSCsG-A-PAA bioconstruct as compared to hADSCsG-A This can be interpreted as a more efficient initiation of thechondrogenic program in the hADSCs condensed clusterswhich were allowed to form in the structured pores of G-A-PAA scaffold as previously discussed Col2a1 synthesisbegan earlier in the hADSCsG-A-PAA system than in thereference system This observation is in accordance to thegeneral findings which state that collagen type II is an earlyvery abundant and highly specific product of differentiatingchondrocytes [67] Under the conditions used in this studyhADSCs revealed an overall greater chondrogenic responsewhen cultivated in the G-A-PAA than in the G-A scaffold asindicated by higher Col2a1 COMP and CEP68 upregulationand more extensive matrix synthesis

Furthermore we simultaneously compared chondro-genic marker expression during 28 days of in vitro chon-drogenesis Heat map analysis (Figure 10(a)) revealed differ-ent patterns of gene expression for the Sox9 chondrogenic

inducer as compared to ECM markers Col2a1 CEP68 andCOMP expressionThe highest levels of gene expression wereregistered for Col2a1 and CEP68 at 28 days postchondro-genic induction in both constructs In contrast Sox9 washighly and constantly expressed between 3 and 14 days ofchondrogenesis but was significantly decreased at day 28 inboth culture systems Thus the heat map analysis confirmedthe distribution of gene expression pattern corresponding toearly and late chondrogenic markers Based on the principlethat genes that form a cluster have similar expression patternwe can predict that in our analysis Col2a1 and CEP68 werethe most related genes together with COMPThis result is inaccordance with the findings of Steck et al 2001 [60] whoconcluded that CEP68 efficiently complements collagen typeII expression in regenerative CTE approaches

Based on this hierarchical agglomerate clustering wefurther analyzed 3D sample distribution for this clusterof cartilage ECM markers and we obtained a graphicalrepresentation in the formof a scatter plot (Figure 10(b))Thisplot displayed information on sample distribution in groupsas a result of the cumulated activity of Col2a1 CEP68 andCOMP genes and highlighted the aligned spatial distributionof their expression in hADSCsG-A and hADSCSG-A-PAApairstime

5 Conclusions

Thecurrent study is a proof-of-concept investigation of a newCTE approach to examine the chondrogenic regenerationpotential of hADSCs and the advantages of natural polymeric3D biomaterials G-A-PAA scaffold proved to better maintainits internal IPN structure and pore shape than G-A control


scaffold probably due to the presence of the synthetic PAApolymer in its formulation Its porous regulated patternallowed hADSCs proliferation in groups of clustered cellswhich favored chondrogenic condensation a critical step inchondrogenesis Despite the presence of PAA in the formu-lation of our sample hADSCs were able to equally adhere toboth G-A-PAA and G-A substrates developing a functionalcytoskeleton and adopting a characteristic spindle-like cellshape Additionally G-A-PAA scaffold supported cellularproliferation and viability and showed no cytotoxic effecton hADSCs confirming its good biocompatibility Takentogether all these features led us to employ our hADSCsG-A-PAA bioconstruct for further differentiation studies Ourchondrogenic differentiation approach revealed that G-A-PAA biomaterial ensured an appropriate microenvironmentfor hADSCs to commit towards the chondrogenic lineageThe chondrogenic markers assessed in our experimentalconditions showed higher levels of expression in hADSCsG-A-PAA as compared to hADSCsG-A biohybrids The iden-tification of collagen type II CEP68 and COMP late chon-drogenic markers suggests that we obtained functional chon-drocytes from hADSCs In conclusion G-A-PAA biomaterialdisplayed an overall adequate profile for in vitro chondrogen-esis approaches which makes hADSCsG-A-PAA biohybridan attractive system for prospective in vivo CTE applications

Conflict of Interests

The authors have declared that no competing interest exists

Authorsrsquo Contribution

Sorina Dinescu and Bianca Galateanu have contributedequally to this work and share the first author position

Acknowledgments

This work was supported by the strategic Grant POS-DRU15915S133391 Project ldquoDoctoral and Post-doctoralprograms of excellence for highly qualified human resourcestraining for research in the field of Life sciences Environmentand Earth Sciencerdquo cofinanced by the European Social Foundwithin the Sectorial Operational Program Human ResourcesDevelopment 2007ndash2013 The costs for consumables andreagents used for this research were supported from Roma-nian CNCS-UEFISCDI Grant no PCCA 1302014 ORALSISThe authors thank Prof Dr Dana Iordachescu (Universityof Bucharest Department of Biochemistry and MolecularBiology) for the project idea and Eugeniu Vasile for the helphe provided with Scanning Electron Microscopy

References

[1] E Steven G Anuhya Z Hilary and B Joel ldquoAttachmentproliferation and chondroinduction ofmesenchymal stem cellson porous chitosan-calcium phosphate scaffoldsrdquo The OpenOrthopedics Journal vol 7 no 1 pp 275ndash281 2013

[2] R Gudas E Stankevicius E Monastyreckiene D Pranys andR J Kalesinskas ldquoOsteochondral autologous transplantation

versus microfracture for the treatment of articular cartilagedefects in the knee joint in athletesrdquo Knee Surgery SportsTraumatology Arthroscopy vol 14 no 9 pp 834ndash842 2006

[3] S B Anderson C-C Lin D V Kuntzler and K S Anseth ldquoTheperformance of humanmesenchymal stem cells encapsulated incell-degradable polymer-peptide hydrogelsrdquo Biomaterials vol32 no 14 pp 3564ndash3574 2011

[4] H L Lim J C Chuang T Tran A Aung G Arya and SVarghese ldquoDynamic electromechanical hydrogel matrices forstem cell culturerdquo Advanced Functional Materials vol 21 no 1pp 55ndash63 2011

[5] J S Park H N Yang D G Woo et al ldquoChondrogenesis ofhuman mesenchymal stem cells mediated by the combinationof SOX trio SOX5 6 and 9 genes complexed with PEI-modifiedPLGA nanoparticlesrdquo Biomaterials vol 32 no 14 pp 3679ndash3688 2011

[6] C Vinatier C Bouffi C Merceron et al ldquoCartilage tissueengineering towards a biomaterial-assisted mesenchymal stemcell therapyrdquo Current Stem Cell Research andTherapy vol 4 no4 pp 318ndash329 2009

[7] P Zuk ldquoAdipose-derived stem cells in tissue regeneration areviewrdquo ISRN Stem Cells vol 2013 Article ID 713959 35 pages2013

[8] M Brittberg A Lindahl A Nilsson C Ohlsson O Isakssonand L Peterson ldquoTreatment of deep cartilage defects in theknee with autologous chondrocyte transplantationrdquo The NewEngland Journal of Medicine vol 331 no 14 pp 889ndash895 1994

[9] A M Bhosale and J B Richardson ldquoArticular cartilagestructure injuries and review of managementrdquo British MedicalBulletin vol 87 no 1 pp 77ndash95 2008

[10] H J Mankin ldquoThe response of articular cartilage tomechanicalinjuryrdquo The Journal of Bone and Joint Surgery Series A vol 64no 3 pp 460ndash466 1982

[11] Y Chen J-Z Shao L-X Xiang X-J Dong and G-R ZhangldquoMesenchymal stem cells a promising candidate in regenerativemedicinerdquo International Journal of Biochemistry and Cell Biol-ogy vol 40 no 5 pp 815ndash820 2008

[12] L Sun M R Reagan and D L Kaplan ldquoRole of cartilage-forming cells in regenerative medicine for cartilage repairrdquoOrthopedic Research and Reviews vol 2 pp 85ndash94 2010

[13] J M Gimble and F Guilak ldquoAdipose-derived adult stemcells isolation characterization and differentiation potentialrdquoCytotherapy vol 5 no 5 pp 362ndash369 2003

[14] D A De Ugarte K Morizono A Elbarbary et al ldquoComparisonof multi-lineage cells from human adipose tissue and bonemarrowrdquo Cells Tissues Organs vol 174 no 3 pp 101ndash109 2003

[15] H A Awad Y-D C Halvorsen J M Gimble and F GuilakldquoEffects of transforming growth factor 1205731 and dexamethasoneon the growth and chondrogenic differentiation of adipose-derived stromal cellsrdquo Tissue Engineering vol 9 no 6 pp 1301ndash1312 2003

[16] F Hildner S Wolbank H Redl M van Griensven and APeterbauer ldquoHow chondrogenic are human umbilical cordmatrix cells A comparison to adipose-derived stem cellsrdquoJournal of Tissue Engineering and Regenerative Medicine vol 4no 3 pp 242ndash245 2010

[17] T G Ebrahimian F Pouzoulet C Squiban et al ldquoCell ther-apy based on adipose tissue-derived stromal cells promotesphysiological and pathological wound healingrdquoArteriosclerosisThrombosis and Vascular Biology vol 29 no 4 pp 503ndash5102009


[18] M W Blanton I Hadad B H Johnstone et al ldquoAdiposestromal cells and platelet-rich plasma therapies synergisticallyincrease revascularization during wound healingrdquo Plastic andReconstructive Surgery vol 123 pp 56Sndash64S 2009

[19] W-S Kim B-S Park J-H Sung et al ldquoWound healing effect ofadipose-derived stem cells a critical role of secretory factors onhuman dermal fibroblastsrdquo Journal of Dermatological Sciencevol 48 no 1 pp 15ndash24 2007

[20] J Rehman D Traktuev J Li et al ldquoSecretion of angiogenicand antiapoptotic factors by human adipose stromal cellsrdquoCirculation vol 109 no 10 pp 1292ndash1298 2004

[21] R B Jakobsen A Shahdadfar F P Reinholt and J E Brinch-mann ldquoChondrogenesis in a hyaluronic acid scaffold compar-ison between chondrocytes and MSC from bone marrow andadipose tissuerdquoKnee Surgery Sports Traumatology Arthroscopyvol 18 no 10 pp 1407ndash1416 2010

[22] I-C Stancu A Lungu D M Dragusin E Vasile C Damianand H Iovu ldquoPorous gelatin-alginate-polyacrylamide scaffoldswith interpenetrating network structure synthesis and charac-terizationrdquo Soft Materials vol 11 no 4 pp 384ndash393 2013

[23] A Lungu M G Albu I C Stancu N M Florea E Vasileand H Iovu ldquoSuperporous collagen-sericin scaffoldsrdquo Journalof Applied Polymer Science vol 127 no 3 pp 2269ndash2279 2013

[24] J A Buckwalter and H J Mankin ldquoArticular cartilage Part Itissue design and chondrocyte-matrix interactionsrdquoThe Journalof Bone and Joint SurgerymdashAmerican Volume vol 79 no 4 pp600ndash611 1997

[25] S M Vickers T Gotterbarm and M Spector ldquoCross-linkingaffects cellular condensation and chondrogenesis in type IIcollagen-GAG Scaffolds seeded with bone marrow-derivedmesenchymal stem cellsrdquo Journal of Orthopaedic Research vol28 no 9 pp 1184ndash1192 2010

[26] L Zheng H S Fan J Sun et al ldquoChondrogenic differentiationofmesenchymal stem cells induced by collagen-based hydrogelan in vivo studyrdquo Journal of Biomedical Materials Research PartA vol 93 no 2 pp 783ndash792 2010

[27] M W Kessler and D A Grande ldquoTissue engineering andcartilagerdquo Organogenesis vol 4 no 1 pp 28ndash32 2008

[28] L Meinel S Hofmann V Karageorgiou et al ldquoEngineeringcartilage-like tissue using human mesenchymal stem cells andsilk protein scaffoldsrdquoBiotechnology and Bioengineering vol 88no 3 pp 379ndash391 2004

[29] J Xu W Wang M Ludeman et al ldquoChondrogenic differenti-ation of human mesenchymal stem cells in three-dimensionalalginate gelsrdquo Tissue Engineering Part A vol 14 no 5 pp 667ndash680 2008

[30] J A Burdick and C Chung ldquoInfluence of three-dimensionalhyaluronic acid microenvironments on mesenchymal stem cellchondrogenesisrdquo Tissue Engineering Part A vol 15 no 2 pp243ndash254 2009

[31] G R Ragetly D J Griffon H-B Lee L P Fredericks WGordon-Evans and Y S Chung ldquoEffect of chitosan scaffoldmicrostructure on mesenchymal stem cell chondrogenesisrdquoActa Biomaterialia vol 6 no 4 pp 1430ndash1436 2010

[32] R L Mauck M A Soltz C C BWang et al ldquoFunctional tissueengineering of articular cartilage through dynamic loadingof chondrocyte-seeded agarose gelsrdquo Journal of BiomechanicalEngineering vol 122 no 3 pp 252ndash260 2000

[33] H Park X Guo J S Temenoff et al ldquoEffect of swelling ratio ofinjectable hydrogel composites on chondrogenic differentiationof encapsulated rabbitmarrowmesenchymal stemcells in vitrordquoBiomacromolecules vol 10 no 3 pp 541ndash546 2009

[34] L Wang I Tran K Seshareddy M L Weiss and M SDetamore ldquoA comparison of human bone marrow-derivedmesenchymal stem cells and human umbilical cord-derivedmesenchymal stromal cells for cartilage tissue engineeringrdquoTissue Engineering Part A vol 15 no 8 pp 2259ndash2266 2009

[35] G Chen D Liu M Tadokoro et al ldquoChondrogenic differ-entiation of human mesenchymal stem cells cultured in acobweb-like biodegradable scaffoldrdquo Biochemical and Biophysi-cal Research Communications vol 322 no 1 pp 50ndash55 2004

[36] H Fan Y Hu C Zhang et al ldquoCartilage regenerationusing mesenchymal stem cells and a PLGA-gelatinchon-droitinhyaluronate hybrid scaffoldrdquo Biomaterials vol 27 no26 pp 4573ndash4580 2006

[37] Y Han Y Wei S Wang and Y Song ldquoCartilage regenerationusing adipose-derived stem cells and the controlled-releasedhybrid microspheresrdquo Joint Bone Spine vol 77 no 1 pp 27ndash312010

[38] F T Moutos B T Estes and F Guilak ldquoMultifunctionalhybrid three-dimensionally woven scaffolds for cartilage tissueengineeringrdquo Macromolecular Bioscience vol 10 no 11 pp1355ndash1364 2010

[39] G Ragetly D J Griffon and Y S Chung ldquoThe effect of type IIcollagen coating of chitosan fibrous scaffolds on mesenchymalstem cell adhesion and chondrogenesisrdquoActa Biomaterialia vol6 no 10 pp 3988ndash3997 2010

[40] SWakitani T Goto S J Pineda et al ldquoMesenchymal cell-basedrepair of large full-thickness defects of articular cartilagerdquo TheJournal of Bone and Joint Surgery Series A vol 76 no 4 pp 579ndash592 1994

[41] J I Huang N Kazmi M M Durbhakula T M Hering J UYoo and B Johnstone ldquoChondrogenic potential of progenitorcells derived from human bone marrow and adipose tissue apatient-matched comparisonrdquo Journal of Orthopaedic Researchvol 23 no 6 pp 1383ndash1389 2005

[42] Y Wei Y Hu W Hao et al ldquoA novel injectable scaffold forcartilage tissue engineering using adipose-derived adult stemcellsrdquo Journal of Orthopaedic Research vol 26 no 1 pp 27ndash332008

[43] X B Jin Y S Sun K Zhang et al ldquoEctopic neocartilageformation from predifferentiated human adipose derived stemcells induced by adenoviral-mediated transfer of hTGF beta2rdquoBiomaterials vol 28 no 19 pp 2994ndash3003 2007

[44] B J Xiao S S Yong K Zhang et al ldquoTissue engineeredcartilage from hTGF 1205732 transduced human adipose derivedstem cells seeded in PLGAalginate compound in vitro and invivordquo Journal of Biomedical Materials Research Part A vol 86no 4 pp 1077ndash1087 2008

[45] H A Awad M Q Wickham H A Leddy J M Gimble andF Guilak ldquoChondrogenic differentiation of adipose-derivedadult stem cells in agarose alginate and gelatin scaffoldsrdquoBiomaterials vol 25 no 16 pp 3211ndash3222 2004

[46] M E Gomes T Rada and R L Reis ldquoAdipose tissue-derivedstem cells and their application in bone and cartilage tissueengineeringrdquo Tissue EngineeringmdashPart B Reviews vol 15 no2 pp 113ndash125 2009

[47] Q ZhaoH Eberspaecher V Lefebvre and BDeCrombruggheldquoParallel expression of Sox9 and Col2a1 in cells undergoingchondrogenesisrdquo Developmental Dynamics vol 209 no 4 pp377ndash386 1997

[48] A M DeLise L Fischer and R S Tuan ldquoCellular interactionsand signaling in cartilage developmentrdquo Osteoarthritis andCartilage vol 8 no 5 pp 309ndash334 2000


[49] D E Bergstrom M Young K H Albrecht and E M EicherldquoRelated function of mouse SOX3 SOX9 and SRY HMGdomains assayed by male sex determinationrdquo Genesis vol 28no 3-4 pp 111ndash124 2000

[50] P Bernard P Tang S Liu P Dewing V R Harley and E VilainldquoDimerization of SOX9 is required for chondrogenesis but notfor sex determinationrdquo Human Molecular Genetics vol 12 no14 pp 1755ndash1765 2003

[51] V LefebvreWHuang V R Harley P N Goodfellow and B DeCrombrugghe ldquoSOX9 is a potent activator of the chondrocyte-specific enhancer of the pro1205721(II) collagen generdquoMolecular andCellular Biology vol 17 no 4 pp 2336ndash2346 1997

[52] D M Bell K K H Leung S C Wheatley et al ldquoSox9 directlyregulates the type-II collagen generdquoNature Genetics vol 16 no2 pp 174ndash178 1997

[53] V Lefebvre R R Behringer and B de Crombrugghe ldquoL-Sox5 Sox6 and SOx9 control essential steps of the chondrocytedifferentiation pathwayrdquo Osteoarthritis and Cartilage A vol 9pp S69ndashS75 2001

[54] M Tsuda S Takahashi Y Takahashi and H Asahara ldquoTran-scriptional co-activators CREB-binding protein and p300 reg-ulate chondrocyte-specific gene expression via association withSox9rdquo The Journal of Biological Chemistry vol 278 no 29 pp27224ndash27229 2003

[55] I Sekiya K Tsuji P Koopman et al ldquoSOX9 enhances aggrecangene promoterenhancer activity and is up-regulated by retinoicacid in a cartilage-derived cell line TC6rdquo The Journal ofBiological Chemistry vol 275 no 15 pp 10738ndash10744 2000

[56] Y Han and V Lefebvre ldquoL-Sox5 and Sox6 drive expression ofthe aggrecan gene in cartilage by securing binding of Sox9 to afar-upstream enhancerrdquoMolecular and Cellular Biology vol 28no 16 pp 4999ndash5013 2008

[57] A Nagy E Kenesi O Rentsendorj et al ldquoEvolutionarily con-served growth plate zone-specific regulation of the matrilin-1promoter L-Sox5Sox6 andNfi factors bound near TATA finelytune activation by Sox9rdquoMolecular and Cellular Biology vol 31no 4 pp 686ndash699 2011

[58] C-J Liu Y Zhang K Xu D Parsons D Alfonso and P EDi Cesare ldquoTranscriptional activation of cartilage oligomericmatrix protein by Sox9 Sox5 and Sox6 transcription factorsand CBPp300 coactivatorsrdquo Frontiers in Bioscience vol 12 no10 pp 3899ndash3910 2007

[59] B Johnstone T M Hering A I Caplan V M Goldberg andJ U Yoo ldquoIn vitro chondrogenesis of bone marrow-derivedmesenchymal progenitor cellsrdquo Experimental Cell Research vol238 no 1 pp 265ndash272 1998

[60] E Steck K Benz H Lorenz M Loew T Gress andW RichterldquoResearch communication chondrocyte expressed protein-68(CEP-68) a novel human marker gene for cultured chondro-cytesrdquo Biochemical Journal vol 353 no 2 pp 169ndash174 2001

[61] P DiCesare ldquoCartilage oligomericmatrix protein (COMP) is anabundant component of tendonrdquo FEBS Letters vol 354 no 2pp 237ndash240 1994

[62] F H Chen M E Herndon N Patel J T Hecht R STuan and J Lawler ldquoInteraction of cartilage oligomeric matrixproteinthrombospondin 5 with aggrecanrdquo Journal of BiologicalChemistry vol 282 no 34 pp 24591ndash24598 2007

[63] H H Mann S Ozbek J Engel M Paulsson and R WagenerldquoInteractions between the cartilage oligomeric matrix proteinand matrilins implications for matrix assembly and the patho-genesis of chondrodysplasiasrdquo Journal of Biological Chemistryvol 279 no 24 pp 25294ndash25298 2004

[64] P E di Cesare F S Chen M Moergelin et al ldquoMatrix-matrix interaction of cartilage oligomeric matrix protein andfibronectinrdquoMatrix Biology vol 21 no 5 pp 461ndash465 2002

[65] K Halasz A Kassner M Morgelin and D Heinegard ldquoCOMPacts as a catalyst in collagen fibrillogenesisrdquo The Journal ofBiological Chemistry vol 282 no 43 pp 31166ndash31173 2007

[66] F H Chen A O Thomas J T Hecht M B Goldring and JLawler ldquoCartilage oligomeric matrix proteinthrombospondin5 supports chondrocyte attachment through interaction withintegrinsrdquo The Journal of Biological Chemistry vol 280 no 38pp 32655ndash32661 2005

[67] HAkiyamaM-CChaboissier J FMartin A Schedl andB deCrombrugghe ldquoThe transcription factor Sox9 has essential rolesin successive steps of the chondrocyte differentiation pathwayand is required for expression of Sox5 and Sox6rdquo Genes andDevelopment vol 16 no 21 pp 2813ndash2828 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


did not prove superior to surgical proceduresThis highlightsthe crucial need to optimize the cell type appropriate scaffoldprochondrogenic factors added in the microenvironmentand the interaction between these components

One of the most difficult tasks in current cell-basedcartilage regenerative therapies is to identify the most suit-able matrix corresponding to each cartilage tissue defectTo develop stable and efficient strategies for CTE severalmaterial design approaches have been investigated [3ndash5]The substrate scaffold should correspond to certain criteriain order to be appropriate for CTE (i) appropriate 3Dstructure in order to recreate the in vivo microenvironment(ii) appropriate porosity to allow cell migration and diffusionof molecules nutrients and oxygen (iii) biocompatibility(iv) biodegradability (v) matrix design to allow its insertionby mini-invasive procedures [6]

Cell types found to be useful in cartilage repair strategiesare represented either by differentiated (chondrocytes) orundifferentiated cells (embryonic stem cells mesenchymalstem cells adipose-derived stem cells [7] synovium-derivedstem cells and periosteum-derived progenitor cells) Iden-tification of the correct cell source is a key aspect for acell-based cartilage reconstruction strategy since these cellsmust exhibit high chondrogenic potential From this pointof view each cell type from stem cells to chondrocytespresents certain advantages and disadvantages leaving themost efficient cell source still debatable

Mature chondrocytes represent possible candidates forcartilage repair due to their ability to produce type II collagensulfated glycosaminoglycans (GAG) and other moleculesspecific for the cartilaginous ECM [8] In the articular carti-lage only 1ndash5 of the volume is occupied by chondrocyteswhich are sparsely spread within the self-secreted ECMmade of collagens proteoglycans and other noncollagenousproteins [9] Considering the above mentioned cell-ECMratio although each chondrocyte has a high activity [10]the overall metabolic rate of the cartilage tissue is low andconsequently conducts to a poor self-regeneration potential

In contrast to primary chondrocytes mesenchymal stemcells (MSCs) are more abundant within certain tissuesmaking themeasier to isolateMain sources forMSC isolationare bone marrow (BM-MSCs) and adipose tissue but MSC-like cells were also identified in other tissue types includingsynovium periosteum skeletal muscle umbilical cord veinor placenta [11] These cells are all capable of chondro-genic differentiation in appropriate culture environments[12] Human adipose-derived stem cells (hADSCs) couldrepresent a viable source of mesenchymal-like stem cells forCTE applications due to their easy harvest [13 14] highchondrogenic potential [13 15 16] and wound healing prop-erties [17ndash20] Despite significant progress obtaining fromhADSCs functional chondrocytes capable of secreting type IIcollagen in tridimensional scaffolds remains challenging [21]

Current regenerative strategies in CTE aim to developappropriate scaffold capable of supporting chondrogenesisThe aim of this study is to evaluate the in vitro biological per-formance of anovel 3D porous scaffold in terms of biocom-patibility and the ability to support hADSCs chondrogenic

differentiation Specifically we developed a tricomponentsystem based on interpenetrating polymer networks (IPNs)of gelatin (G) alginate (A) and polyacrylamide (PAA) Westudied the evolution of prochondrogenic induced hADSCscommitment towards functional chondrocytes inside this 3Dporous scaffold which proved to be biocompatible and topossess physical and chemical properties similar to cartilageThis work contributes to current efforts to address thechallenges of using smart biomatrices to unleash the potentialof stem cells for curative CTE

2 Materials and Methods

21 Scaffold Synthesis Gelatin B (G) from bovine skin(Sigma) was used as 20 (wv) aqueous solution Sodiumalginate (SA) was used as 4 (wv) aqueous solution Acryl-amide (AA) for electrophoresis gt99 (HPLC) and NN1015840-methylenebis(acrylamide) (MBA) 99 triethanolamine(TEA) ammonium persulphate (APS) glutaric aldehyde(GA) as aqueous solution 25 and calcium chloride anhy-drous (CaCl

2) were purchased from Sigma and used without

further purificationIPNs based on G calcium alginate (A) and polyacry-

lamide (PAA) were prepared using a three-step procedurereported elsewhere [22] The three components were used ina weight ratio of 14 1 20 between G SA and AA with atotal solid content (T) of 21 Initially cross linked PAA(molar ratio between MBA and AA was 18 100) was gen-erated through network-forming radical polymerization ofsynthetic monomers in the presence of the natural polymersRedox initiation based on APS (1 molar with respect toAA and MBA) and TEA (12 molar with respect to APS)was used to perform the polymerization reaction undermild conditions Briefly the polymerization stock solutionwas prepared based on a protocol detailed elsewhere [22]through the dissolution of AA MBA and the correspondingamount of initiator in distilled water under stirring at roomtemperature (RT) 1mL of this solution was further mixedwith 8mL of gel solution and with 1mL of SA solution at40∘C The resulting viscous solution was degassed using anultrasound bath (Elma S 30H Elmasonic) for 15 minutes at40∘CThe polymerization accelerator TEA was added undervigorous stirring The network-forming polymerization ofAA and MBA was allowed to occur in Petri dishes for 24hours at 40∘C Semi-IPNs consisting in cross-linked PAAand uncross-linked G and SA were obtained Then thematerials were cooled for 2 hours at 4∘C to allow physicalgelation of G G was cross-linked through immersion in GA05 for 24 hours at RT followed by the cross-linking ofSA in a 1 CaCl

2aqueous solution for 24 hours Calcium

alginate (A) is thus formedThe resulting G-A-PAA hydrogelwas further purified in distilled water at 40∘C for four daysGravimetric measurement was used to assess the successof the IPNs formation through gel fraction (GF) analysisThe experiment consists in the assessment of GF as rationbetween remaining gel (dried) after extensive extraction ofthe eventual soluble fraction in distilled water at 40∘C with



GF =119898gel

1198980

times 100 (1)






























(a) (b)

(c) (d)




3 Results






(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)






50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



GF =119898gel

1198980

times 100 (1)






























(a) (b)

(c) (d)




3 Results






(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)






50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















(a) (b)

(c) (d)




3 Results






(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)






50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)




3 Results






(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)






50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a1) (a2) (a3)

(b1) (b2) (b3)

(a5) (a6)(a4)

(b4) (b5) (b6)






50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)









(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a1)

(a2)

(a3)

(b1)

(b2)

(b3)

(c1)

(c2)

(c3)

100120583m 100120583m 100120583m

100120583m100120583m100120583m

100120583m 100120583m 100120583m


xy z

8188 120583m000 120583m


(a)

10808 120583m

000 120583m

xy z


(b)






00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00

01

02

03

04

05

06

Abso

rban

ce at

550

nmMTT assay


hADSCsPAA

lowastlowastlowast

lowast

24h 5d 7d

(a)

00

01

02

03

04

05

Abso

rban

ce at

490

nm

G-A-PAAG-A

PAA

24h 5d 7d

LDH assay

lowast

(b)
























T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


T0 3d 7d 14d 28d


00

05

10

15

Fold

incr

ease

Sox9

lowastlowast

lowast

lowastlowast

∙∙∙

∙∙∙

(a)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowastlowastlowast


Fold

incr

ease

0

1

2

3

4

5

Col2a1

∙∙∙

(b)

0

1

2

3

4

5

Fold

incr

ease

CEP68

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


∙∙

(c)

0

1

2

3

4

Fold

incr

ease

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowast

lowast


COMP

∙∙∙

(d)






(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

100120583m

100120583m

100120583m 100120583m

100120583m100120583m

100120583m

100120583m




0

10

20

30

Con

cent

ratio

n (n

gm

L)

T0 3d 7d 14d 28d

lowastlowastlowast

lowastlowastlowast

lowast


COMP

∙∙∙

∙∙


4 Discussion















1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

2

3

4

5

6

7

8

9

10

11

28d

hAD

SCs

G-A

-PA

A28

d hA

DSC

sG

-A14

d hA

DSC

sG

-A-P

AA

14d

hAD

SCs

G-A

7d

hAD

SCs

G-A

-PA

A7

d hA

DSC

sG

-A3

d hA

DSC

sG

-A-P

AA

3d

hAD

SCs

G-A

T0

hAD

SCs

G-A

-PA

AT0

hAD

SCs

G-A

Col2a1

CEP68

COMP

Sox9

(a)

0

0

1 2 3 4 5 6 7 8 9 10

10

11

0

1

2

3

4

5

6

7

8

9

10

11



(b)







5 Conclusions








Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Acknowledgments


References














































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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