Chondrogenic potential of injectable k-carrageenanhydrogel with encapsulated adipose stem cells forcartilage tissue-engineering applicationsElena G. Popa1,2, Sofia G. Caridade1,2, João F. Mano1,2, Rui L. Reis1,2 and Manuela E. Gomes1,2*13Bs Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães, Portugal2ICVS/3Bs – PT Government Associate Laboratory, Braga/Guimarães, Portugal

Abstract

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatmentof cartilage defects must use a significant amount of cells, preferably applied using a hydrogel systemthat can promise their delivery and functionality at the specific site. This paper discusses the potentialuse of k-carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in thetreatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropicgelationmethod and human adipose stem cells (hASCs) were encapsulated in 1.5%w/v k-carrageenansolution at a cell density of 5�106 cells/ml. The results from the analysis of the cell-encapsulatinghydrogels, cultured for up to 21days, indicated that k-carrageenan hydrogels support the viability,proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysisdemonstrated an increase in stiffness and viscoelastic properties of k-carrageenan gels with theirencapsulated cells with increasing time in culture with chondrogenic medium. These results allowedthe conclusion that k-carrageenan exhibits properties that enable the in vitro functionality ofencapsulated hASCs and thus may provide the basis for new successful approaches for the treatmentof cartilage defects. Copyright © 2013 John Wiley & Sons, Ltd.

Received 10 October 2012; Accepted 14 November 2012

Keywords adipose-derived stem cells; cartilage; chondrogenic differentiation; hydrogels; k-carrageenan;mechanical properties

1. Introduction

The need for tissue-engineered cartilage constructs is im-mense and of great clinical significance, because no existingmedication or surgical procedures substantially promotesthe healing process of articular cartilage. Compared to otherconnective tissues, cartilage is an avascular, aneural tissue,consisting of relatively few cells, proteoglycans andproteins, and thus, when damaged due to a degenerativedisease or trauma, the functional and the metabolicproperties of the original hyaline tissue will hardly ever berestored (Redman et al., 2005). Cartilage tissue-engineeringapproaches, adopting the delivery of an appropriate type

and amount of cells, with or without signalling factors, offerconsiderable promise as regeneration strategies (Tuli et al.,2003; Nesic et al., 2006). However, for further improve-ment, minimally invasive approaches and innovative cell-carrier concepts should be refined, using the rightbiomaterials and cell sources (Sittinger et al., 2004). Stemcells are the best chance for human cartilage regeneration,given their ability to differentiate in vitro in differentlineages, availability and expansion (Vallee et al., 2009).In particular, adipose tissue has generated significant inter-est in cartilage tissue engineering as an abundant source ofmultipotent progenitor cells, which are easily acquired athigh yields and can be obtained from the patient by mini-mally invasive procedures, such as liposuction (Ericksonet al., 2002; Augst et al., 2006; Tapp et al., 2009). Hydrogelsare the basis of cell-encapsulation systems, one of the mostpromising approaches for the delivery of cells and therapeu-tic agents to the site of interest (Jen et al., 1996; Drury andMooney, 2003; Slaughter et al., 2009). Different hydrogels

*Correspondence to: M. E. Gomes, 3Bs Research Group –

Biomaterials, Biodegradables and Biomimetics, University ofMinho, Headquarters of the European Institute of Excellenceon Tissue Engineering and Regenerative Medicine, Guimarães4806-909, Portugal. E-mail: megomes@dep.uminho.pt

Copyright © 2013 John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLEJ Tissue Eng Regen Med (2013)Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1683

have previously been studied for biomedical applications,such as alginate and agarose (Augst et al., 2006; Varoniet al., 2012). In the present study we evaluated the potentialuse of k-carrageenan hydrogel extracted from red algae as acell carrier system. k-Carrageenan is an ionic naturalpolysaccharide that displays unique properties that mayprovide advantageous features for application in thetissue-engineering field. Carrageenan is composed of large,highly flexible molecules which curl, forming helicalstructures (Bixler, 1994; Sipahigil and Dortunc, 2001),rendering them able to form a variety of different gels atroom temperature and thus enabling great versatility. Thefully carbohydrate-based hydrogels of alternating copol-ymers of a-(1-3)-D-galactose and b-(1-4)-3,6-anhydro-D-or L-galactose (Meunier et al., 2001) are expected tobe biologically compatible, biodegradable and non-toxic.k-Carrageenan has only one negative charge per disaccharide,with a tendency to form a strong and rigid gel. Carrageenangels in the presence of potassium ions but also has the abil-ity to form gels under salt-free conditions, being a thermo-sensitive hydrogel (Lennart, 2006). The mechanism of gelformation is still under discussion; nevertheless, the firststep may well be double helix stabilization when K+ is thecounter-ion (Kara et al., 2003). Because of the ionicnature of the polymer, the gelling andmelting temperaturesof k-carrageenan are almost solely dependent on the con-centration of potassium ions (Mangione et al., 2005). Ther-moreversible gels, such as k-carrageenan, melt at elevatedtemperatures, and lowering the temperature results in thegelation of the biopolymer. The temperature-induced gela-tion allows for the easy formation of gels in different shapes.The negative charge permits ionic interactions with mole-cules such as water or protein and allows a large osmoticswelling pressure. To some extent, k-carrageenan resemblesnaturally occurring glycosaminoglycans, owing to theirbackbone composition of sulphated disaccharides(Velleman, 1999). At present there is little informationabout the potential use of k-carrageenan as a new naturalmaterial in biomedical applications. Nevertheless, theresults published to date suggest that k-carrageenan hydro-gels exhibit comparable biological behaviour to, but highermechanical properties than, similar hydrogels extensivelyused for tissue-engineering purposes (Awad et al., 2004;Bosnakovski et al., 2006; Kisiday et al., 2008). The objectiveof this study was to develop k-carrageenan hydrogel aimedat application in cartilage tissue engineering. Therefore, theswelling and cytotoxicity of the hydrogels were studied, aswell as their ability to sustain the proliferation and chondro-genic differentiation of hASCs. Furthermore, the mechani-cal properties of hydrogels with encapsulated hASCs afterdifferent times in culture were also evaluated. The resultsobtained demonstrated that k-carrageenan hydrogels arenon-cytotoxic and support the proliferation and chondro-genic differentiation of encapsulated hASCs. Additionally,the mechanical properties of the cell-loaded hydrogels aremaintained and even increased with increasing culturetime, suggesting that the proposed systems provides apromising alternative to current approaches for cartilagetissue engineering.

2. Materials and methods

2.1. k-Carrageenan hydrogel preparation

An aqueous solution was prepared by dissolving thek-carrageenan powder (Sigma Fluka) in distilled water,and heating at 60 �C while stirring constantly for 2 h untilcomplete and homogeneous dispersion of the materialwas obtained. Previous to use, the k-carrageenan solutionwas autoclaved for 30min at 120 �C and prepared justbefore use. k-Carrageenan hydrogel samples were producedusing the sterilized polymeric solution with a final concen-tration of 1.5% w/v and 5% w/v potassium chloride (KCl;Sigma) as a crosslinker reagent. The samples were shapedin the form of discs, using cylindrical moulds, and a com-plete gelation carried out for 2–5min at room temperatureto form a solid gel. After gelation, the moulds’ ends werecut and the cylindrical hydrogels pushed out andimmersed in KCl for 15–30min in order to stabilize thethree-dimensional (3D) structure. Afterwards, the gelswere washed with phosphate-buffered saline (PBS;Sigma) to remove the excess KCl present in the materials.Sample discs were then sliced off the hydrogel cylinders,using a thin sterile blade; disc dimensions were diameter5� 0.01�height 2.5� 0.46mm. These samples wereused for evaluating the swelling kinetics and cytotoxicityof the hydrogel.

2.2. k-Carrageenan hydrogel physicalcharacterization

2.2.1. Swelling kinetics

The swelling kinetics of the developed hydrogels wasstudied in a PBS solution (different pH values weretested) and in culture medium [Dulbecco’s modifiedEagle’s medium (DMEM), supplemented with fetal bovineserum (FBS; Gibco]. For this purpose, a weighed amountof each hydrogel formulation was immersed in eachtesting solution (PBS and DMEM, pH 7.4) and incubatedat 37 �C under static conditions. The influence of pH ofthe PBS solution on the water uptake of k-carrageenanhydrogel was also analysed. The swelling ratio wascalculated using Equation 1:

% Equilibrium swelling ratio ESRð Þ ¼ Ws �Wd

Wd� 100

(1)

where Ws is the weight of swollen gel after reachingequilibrium value under specified environmental conditions(PBS or DMEM) and Wd is the weight of the dried gel. Tomeasure Ws, the swollen hydrogels were removed fromthe PBS or DMEM and immediately weighed with a micro-balance after the excess of water lying on the surfaces wasabsorbed with a filter paper. Three samples were used foreach testing condition (n=3).

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Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

2.3. k-Carrageenan hydrogel biologicalevaluation

2.3.1. Cytotoxicity screening

The cytotoxicity of the leachable hydrogels was evaluatedusing L929 mouse fibroblast cells [European Collection ofCell Cultures (ECACC), UK], as described elsewhere(Gomes et al., 2001). Extracts of the study materials wereprepared and placed in contact with a L929 cells mono-layer. Briefly, the extracts were obtained after 24 h incuba-tion of the hydrogels in complete culture medium at 37 �Cand under constant agitation (60 rpm). The ratio of thehydrogel to the extract fluid was 3 cm2/ml and the L929cell-seeding density was 4�103 cells/well plate. In allcytotoxicity tests performed, latex rubber was used asthe positive control for cell death and standard tissue cul-ture polystyrene was used as a negative control. Theobjective of the extraction test was to evaluate changesin cell morphology and growth inhibition, whereas theMTS (CellTiter 96W Aqueous One Solution Cell Prolifera-tion Assay, G3580, Promega) test determines whethercells are metabolically active (Salgado et al., 2004). Theoptical density (OD) was read at 490 nm on a multiwellmicroplate reader (Synergie Ht Izasa, Bio-Tek Instruments)and all cytotoxicity screening tests used six replicates.

2.3.2. Human adipose-derived stem cellsisolation and expansion

Human liposuction aspirate samples were obtained fol-lowing informed consent from donors undergoing lipoas-piration procedures under a protocol established withthe Department of Plastic Surgery of the Hospital daPrelada, Porto, and approved by the local Ethical Committee.All the samples were processed within 24h after collection.Human adipose-derived stem cells (hASCs) were enzymati-cally isolated as previously described (Rada et al., 2010).Briefly, the adipose tissue samples were digested with 0.2%collagenase type II (Sigma) in PBS for 45min at 37 �C undergentle stirring. The digested tissue was filtered with a100mm filter mesh (Sigma) centrifuged at 1200 rpm for10min at 20 �C, and the cell suspension solution washedfor 5min with lysis buffer to remove erythrocytes. The cellswere again centrifuged and the supernatant removed andresuspended in a-minimal essential medium (a-MEM;Gibco) with 10% FBS (Gibco; heat-inactivated), 1% antibi-otic–antimycotic (Invitrogen) and sodium bicarbonate(NaHCO3; Sigma). Human ASCs were plated at a densityof 3.5�103 cells/cm2 and incubated at 37 �C in a humidifiedatmosphere of 5% CO2. The unattached cells were removedafter 2–3days with repeated PBS washings. The adherenthASCs were cultured with medium changes every 3days.Confluent cultures were passaged with 0.05% trypsin(Invitrogen). The enzymatic treatment was quenched inthe presence of FBS and cell counts were done using ahaemocytometer. Only passage 3 (P3) hASCs were used inthe experiments.

For the cell culture experiments, adipose-derived stemcells were isolated from one patient (a women aged31 years) and cryopreserved at passage 1, to avoid patientcells variability. Afterwards, the cells were expanded andused until P3, performing three independent experimentsusing the same batch of cells, with the same passage. Thephenotype of hASCs has been extensively investigated inprevious studies, using tissue sample from the sameanatomical site (subcutaneous), harvested with a similartechnique (such as lipoaspiration) and isolated followingsimilar procedures (digestion with collagenase) byZuk et al. (2002) and Gimble et al. (2007) and alsoby our research group (Carvalho et al., 2011a, 2011b;Rada et al., 2011).

2.3.3. Encapsulation of hASCs in k-carrageenanhydrogels and in vitro cell culture

k-Carrageenan aqueous dispersions were prepared justbefore use by dissolving the biopolymer in distilled waterand then sterilizing, as described in previous section.Human ASCs were detached by trypsin and centrifugedat 200� g for 7min. The cells were resuspended in sterilePBS solution, counted using a haemocytometer and finallycentrifuged. The supernatant was discarded and k-carra-geenan 1.5% w/v solution was added to the cells to obtaina final concentration of 5�106 cells/ml. The mixture wasresuspended for complete homogenization of the cellswithin the matrix. Hydrogel samples containing humanASCs were prepared using sterile cylindrical moulds andallowing to rest at room temperature for 1–2min to form asolid gel. Discs of 5� 0.01mm diameter and 2.5� 0.46mmheight were cut using a sterile blade. The discs with encapsu-lated cells were cultured in either basal or chondrogenicdifferentiation medium for 1, 7, 14 and 21days, with the me-dium replaced every 3–4days. Additional controls consistedof k-carrageenan hydrogel samples without cells, kept underthe same culture conditions for the selected time periods.The chondrogenic differentiation medium was composed oflow-glucose DMEM (Sigma) supplemented with 10% FBS(Gibco), 1% antibiotic–antimycotic (Gibco), ITS+1 LiquidMedia Supplement (I2521; insulin–transferrin–seleniumliquid media supplement, Sigma), 17mML-ascorbic acid(Sigma), 0.1M sodium pyruvate (Sigma), 35mML-proline(Sigma), 1mM dexamethasone (Sigma) and 10ng/mlhuman transforming growth factor-b1 (TGFb1; eBioscience).

2.4. k-Carrageenan hydrogel biologicalcharacterization

2.4.1. Cell viability and proliferation assessment

At the end of each time point of the study, the k-carrageenansamples with encapsulated hASCs were collected from theculture plates and incubated in a Calcein AM (Invitrogen)solution of 1/1000 in culture medium for 15–30min at37 �C and afterwards washed in sterile PBS. The stainedsamples were placed on a microscope slide and observed

Chondrogenic potential of injectable k-carrageenan hydrogel

Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

under fluorescent microscopy (reflected/transmitted lightmicroscope; Zeiss). The proliferation of the hASCs encapsu-lated in the k-carrageenan hydrogels was assessed usinga fluorimetric double-strand DNA quantification kit(PicoGreen, Molecular Probes). For this purpose, samplescollected at 1, 7, 14 and 21days of culture were washed inPBS and then transferred into 1.5ml microtubes containing1ml ultrapure water. Prior to dsDNA quantification, hASC–hydrogel constructs and the sample controls (hydrogelsamples without cells) were sonicated for 15min to releaseall DNA from the hydrogel. Samples and standards(0–2mg/ml) were prepared and mixed with a PicoGreensolution in a 200:1 ratio and added to a 96-well opaquewhite plate. For each study material and standard, threesamples were used for DNA assays, and triplicates of eachsample were measured; the procedure followed can befound elsewhere (Labarca and Paigen,1980) andwas basedon themanufacturer’s instructions. The plate was incubatedfor 10min in the dark and fluorescence was measured on amicroplate ELISA reader (Bio-Tek, Synergie HT) with anexcitation of 485/20nm and an emission of 528/20nm. Astandard curve was created, and sample DNA values wereread from the standard graph.

2.4.2. Histological analysis

Samples were collected at the end of the experiment(21 days of culture) fixed, dehydrated with a Spin TissueProcessor (Microm STP120 Inopat) and embedded in par-affin using an embedding centre (Microm EC350-1/EC350-2 Inopat). Sections were cut at 4 mm with a micro-tome (Microm HM355S Inopat) and placed on micros-copy slides. Alcian blue (Sigma), safranin O (Sigma),toluidine blue (Sigma) and haematoxylin and eosin(H&E; Sigma) stainings were performed using the Auto-matic Stainer equipment (Microm HMS740 Inopat). Alcianblue, safranin O and toluidine blue were used to evaluatecartilage ECM components (glycosaminoglycans) deposi-tion. Alcian blue staining was performed by rinsing the sec-tions in 3% acetic acid (Sigma) and incubating them in 1%alcian blue solution for 30min. After that, the stain waspoured off and the sections counterstained with aqueousneutral red (Sigma) for 1min, then dehydrated. SafraninO staining consisted of staining the sections with Weigert’siron haematoxylin working solution for 7min, fast green(Sigma) for 5min and 0.1% safranin O for 5min. The sec-tions were washed after each staining step, left to dry in airand then rinsed in absolute alcohol. Toluidine blue stainingsolution was prepared by dissolving 1% toluidine blue indistilled water containing 0.5 g sodium borate (Riedel-de-Haën), followed by filtering, and the sections weredipped in for 2–3 s. For H&E staining, after hydrationthe sample sections were coloured with PapanicolaouHarris haematoxylin (Bio-optica) for 3min, washed inrunning tap water and afterwards a blue stain enhance-ment was performed by immersion in 0.5% ammonia(Sigma) for 5–10 s. The sections were then washed inrunning tap water and stained in eosin-Y (Bio-optica)for 30 s. Finally all slides were dehydrated by immersion

in a series of alcohols (30–100%). The final step forall stainings was immersion in the clearing agentHistoclearW (National Diagnostics) or xylene substitutefor 1–2min, then mounting using Microscopy EntellanW

(Merck) for later observation. The stained sections wereobserved under a light microscope (reflected/transmit-ted light microscope; Zeiss).

2.4.3. Immunohistochemical analysis

Sections of samples corresponding to all experimentalconditions were obtained as described above. Beforeremoving the paraffin the slides were warmed, and anti-gen retrieval was performed for 20min at 95 �C using10mM citrate buffer. The sections were washed in PBSfor 10–15min and endogenous peroxidase activity wasquenched with 3% hydrogen peroxide (Sigma) in 50%methanol/tap water for 5min. Afterwards, the sampleswere washed with PBS and blocked with 3% bovineserum albumin (BSA; Sigma) for 1 h to avoid non-specificstaining. The sections were further incubated with primaryantibodies [collagen types I and II; mouse antitype II colla-gen (MAB1330) andmouse antitype I collagen (MAB3391);Chemicon) overnight at 4 �C in a humidified atmosphere.Then the slides were washed with PBS for 10min and incu-bated with secondary antibody from a Vectastain Elite ABCKit (Vector Laboratories, Peterborough, UK) for 1 h at roomtemperature, again in a humidified atmosphere. The remain-ing protocol was performed according to that described inthe in the Vector DAB Kit (Vector Laboratories). The slideswere washed in water for 5min and then counterstainedwith haematoxylin for nuclei visualization, and finallymounted. Controls were performed using normal horseserum replacing the primary antibodies, which was alsoincluded in the kit. The samples were visualized undera light microscope and images obtained using a camera(Axion MRc5, Zeiss).

2.4.4. RNA isolation and real-time quantitativeRT–PCR

Real-time qRT–PCR analysis was used to assess theexpression profile of typical markers for chondrogenicdifferentiation, namely Sox9, Aggrecan, Collagen X,Collagen type I and Collagen type II, and thereby to evalu-ate the ability of hASCs to undergo differentiation whenencapsulated in k-carrageenan and cultured with eitherbasal or chondrogenic medium. For this purpose, totalRNA was extracted from cell–hydrogel constructs usingTRI ReagentW RNA Isolation Reagent (Sigma) accordingto the technical data sheet provided. Briefly, three sam-ples of each condition were collected at defined time per-iods, washed twice with PBS, added to 800 ml TRI Reagentand vigorously mixed for 10 s, then stored at –80 �C untilthe analysis was performed. At this point, 160 ml chloro-form was added and the samples incubated on ice for15min and centrifuged at 13 000 rpm for 15min at 4 �Cto establish a three-phase composition in the tube. Theaqueous phase was collected into new, clean, prechilled

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Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013)DOI: 10.1002/term

tubes, where 400 ml ice-cold isopropanol was added, andthe samples were incubated at –20 �C overnight. Thesamples were then centrifuged at 13 000 rpm for 15minat 4 �C, the supernatant discarded and the pellet washedwith 70% ethanol. After a final centrifugation, the sam-ples were allowed to dry in air and suspended in ultrapurewater for posterior analysis. Each pellet was dissolved in15ml RNase-free water and kept at –80 �C until use. Theamount of isolated RNA and A260/280 nm ratio were quan-tified using a Nanodrop ND-1000 spectrophotometer(Bonsai 06/2008 NanoDrop Technologies, Wilmington,DE, USA). After these determinations, 2 mg RNA of eachsample was reverse-transcribed using a qScript™ cDNASynthesis Kit (Quanta Biosciences) in a 40 ml reaction,using a MJ Mini™ Personal Thermal Cycler (Bio-RadLaboratories) machine. Real-Time qRT–PCR was performedto detect amplification variations, using PerfeCTaW

SYBRW Green FastMixW (Quanta Biosciences) on anEppendorf MastercyclerW ep realplex gradient S machine.The analysis of the results was performed with realplexsoftware (Eppendorf Mastercycler, Applied Biosystems).The reaction composition was as follows: 10ml SYBR GreenPCR FastMix, 2.5ml each forward and reverse primer(see Table 1) and 5ml diluted template with RNase-freewater. The number of cycles and annealing temperaturewere selected according to the manufacturer’s instructions.All the primer sequences were generated using Primer3software v 0.4.0 and acquired from Eurofins MWG Operon(Ebersberg). More details can be found in Table 1. Eachgene was processed in triplicate. Human ASCs encapsu-lated in k-carrageenan hydrogel cultured in basal mediumwere used as control cell sample. In each sample the mRNAlevel expression of each gene was normalized to theaverage expression of GAPDH value. The relative geneexpression quantification was performed using the 2–ΔCT

and 2–ΔΔCT methods (Livak and Schmittgen, 2001). Threesamples of each material/conditions under study wereanalysed.

2.4.5. Mechanical properties of k-carrageenanhydrogels with encapsulated hASCs

Dynamic mechanical analysis (DMA; Tritec 2000 Triton)was conducted to characterize the mechanical properties,under compression loading, of k-carrageenan hydrogelsamples and hydrogels samples with encapsulated hASCs,

after culture in either chondrogenic or basal medium fordifferent time points. The samples were prepared, asdescribed above, into discs of 8� 0.01mm diameter and2.5� 0.38mm height. The study samples (n= 5) weresubjected to compression cycles of increasing frequencyin the range 0.1–10Hz, with constant amplitude displace-ments of 0.1mm, using DMA equipment (Triton Technol-ogy, Nottinghamshire, UK). Experiments were performedunder simulated physiological conditions at 37 �C in PBSmedium. The frequency scans, with acquisitions of 15points/decade, were performed at 37 �C with a heatingrate of 2 �C/min. The mechanical analysis results werepresented in terms of two main parameters: storage mod-ulus (E0; the in-phase, elastic component) and loss modu-lus (E00; the out-of-phase, viscous component). The valuesfor the compression modulus were collected at a fre-quency of 0.1–10Hz along the 21 days of culture, anddamping factor was expressed for samples cultured inchondrogenic medium.

2.5. Statistical analysis

Data obtained from DNA and real-time qRT–PCR analysisare presented as mean� standard deviations (SD; n= 3).First, a Shapiro–Wilk test was used to ascertain the datanormality. DMA data were first inspected with an F-testfor treatments, to determine equality of variance (Liski,2009). The results indicated that at the 0.05 level, thedata were significantly drawn from a normally distributedpopulation, and one-way ANOVA followed by Tukey testwas used to determine significant differences betweengroups and conditions, using with OriginPro 8 program.

3. Results and discussion

3.1. Swelling kinetics of k-carrageenan hydrogel

The k-carrageenan hydrogels were obtained by a mild cross-linking reaction with K+ ions at a stable physiological pH.The influence of phosphate buffer solution pH on the swell-ing properties of k-carrageenan hydrogels was analysed,determining the equilibrium swelling at pH 1, 4, 7.4 and 11as a function of time (Figure 1). These results showed thatswelling increases with time, reaching a constant maximum

Table 1. List of genes under evaluation, primers and annealing temperatures used in the analysis of samples corresponding to en-capsulated hASCs in k-carrageenan and cultured in vitro for different time points

Target gene1 Forward sequence Reverse sequence Tm (�C)

SOX9 50-tacgactacaccgaccacca-30 50-ttaggatcatctcggccatc-30 58.4Aggrecan 50-tgagtcctcaagcctcctgt-30 50-tggtctgcagcagttgattc-30 58.4Collagen X 50-ccaggtctcgatggtcctaa-30 50-gtcctccaactccaggatca-30 59.4Collagen I 50-catctccccttcgtttttga-30 50-ccaaatccgatgtttctgct-30 55.3Collagen II 50-gacaatctggctcccaac-30 50-acagtcttgccccacttac-30 56.4GAPDH 50-acagtcagccgcatcttctt-30 50-acgaccaaatccgttgactc-30 57.3

1Real-time qRT–PCR thermal cycles correspond to the following periods: 2min at 95 �C (hot start), followed by 45 cycles of 95 �C for 30 s,corresponding annealing temperature (noted in the table) for 30 s and an extension step at 68 �C for 30 s. A melting curve of 21min and ahold step at 5 �C was performed at the end.

Chondrogenic potential of injectable k-carrageenan hydrogel

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value after 48h. In general, it was observed that the swellingratio increasedwith increasing pH values of the PBS solution,except for pH 11, which registered the lowest value for thefirst 28h post-immersion. The amount of water absorbedduring the first 7h in PBS at pH 7.4 registered the highestvalues, showing that the developed hydrogels exhibit thehighest water uptake at physiological pH. The swelling kinet-ics of k-carrageenan hydrogels were also investigated whenimmersed in cell culture medium (DMEM, pH 7.4, at 37 �C)supplemented with 10% serum (FBS) and compared withPBS (pH 7.4, at 37 �C), as the hydrogels will eventually beexposed to this medium during cell encapsulation and cul-ture (Figure 1). It could be observed that the swelling ratioin DMEM/10% FBSmediumwas lower than in PBS solution.

It is known that, upon implantation of a biomaterial inthe body, a response typically occurs that is characterizedby a decrease in the local pH. Thus, it was thought itwould be important to know the behaviour of the hydro-gel at various pHs in order to predict and eventually tailorits response upon implantation, envisioning in vivo studiesand possible future clinical applications. When increasingthe pH values of the immersion solutions from 1 to valuesequal to the physiological pH, a significant increase ofswelling ratio leading to high water absorption wasobserved (Figure 1). The k-carrageenan hydrogels soakedin medium at pH 11 presented lower water uptake,revealing that the equilibrium swelling was dependentupon pH and the ionic composition of the immersionmedia. As the pH of the PBS solution was> 4, the ioniza-tion of the sulphated groups of k-carrageenan geloccurred and led to ionic repulsion. This resulted in amore hydrophilic polymer network and contributed tohigher water absorption. In summary, the swelling resultsdemonstrated that the hydrogels exhibited a pH-dependentpattern in the range of pH studied. Furthermore, theseresults support that the amount of ions present in themedium significantly affects the swelling properties of thehydrogels, as confirmed by the different results obtained

upon immersion in PBS and DMEM media (Naim et al.,2004). It is important to note that ionically crosslinkedk-carrageenan loses stability over time in vitro, mostprobably due to an outward flux of crosslinking ions intothe surrounding medium (Shoichet et al., 1996).

3.2. Cytotoxicity assessment

The cell behaviour of hydrogel extracts was studied as apreliminary approach to testing the in vitro toxicity ofthe developed k-carrageenan hydrogels. The cytotoxicityof k-carrageenan hydrogels was evaluated by culturingL929 cells for 1 and 3 days with extracts of the developedmaterial. Figure 2 shows the results of cell metabolic

Figure 1. Equilibrium swelling ratio of k-carrageenan hydrogels as a function of time, registered after immersion in PBS solution atdifferent pH (1, 4, 7.4, 11) and after immersion in DMEM/10% FBS at pH 7.4 and 37 �C. Values reported as averages (n=3)�SD

Figure 2. Graphical representation of L929 cells metabolic activ-ity after incubation with extracts of 1.5% w/v k-carrageenanhydrogel, TCP (negative control) and latex (positive control)evaluated by MTS assay. *Significantly higher metabolic activityat day 3 as compared to values obtained at day 1 of culture.Statistical analyses were conducted using one-way ANOVA forn=6 and error bars represent mean�SD

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activity assessed by the MTS test performed with hydrogelextracts. It can be observed that the viability levels weresimilar to the values obtained for the negative control(TCPs), evidencing that k-carrageenan and possibleleachable products did not exert any cytotoxicity effecton L929 cells. A significant increase in cell viabilitywas observed from day 1 to the last time point of the assay(*p< 0.05). The cytotoxicity assessment of k-carrageenanextracts was carried out as a preliminary approach toassessing the potentially harmful effect of the developedhydrogels. The cytotoxicity results (Figure 2) indicatedthat the cells were viable in the presence of hydrogelextracts for all the time points assessed. The cells incontact with the hydrogel extracts recorded levels ofmetabolic activity similar to those which were in contactwith culture medium, showing extremely low cytotoxicitylevels, even after long exposure.

3.3. Fluorescence staining and DNAquantification

Fluorescence staining was conducted to analyse theviability of the cells after encapsulation/culture on 1.5%w/v k-carrageenan hydrogels (Figure 3A). Calcein AM

confirmed the viability of hASCs after in vitro culture,establishing that the cells were viable and homogeneouslydistributed inside the hydrogels. Three weeks post-encapsulation, a high density of cells was observed andthe cells were viable, exhibiting a round shape within thehydrogel matrix. These results confirmed that thetemperature cycle used to promote the sol–gel transitionof k-carrageenan did not affect cell viability.

Complementary to the viability analysis along thecourse of the experiment, the cell content was quantifiedbased on dsDNA quantification (Figure 3B). Analysis ofobtained DNA values confirmed that from day 1 to day 7the cell number slightly decreased for the two conditions,followed by a significant increase from day 14 to day 21(*p< 0.05 for basal medium and **p< 0.05 for chondro-genic medium). These results are also in agreement withthe fluorescence viability assay (Figure 3A), showing thatthe k-carrageenan hydrogel enables the viability andproliferation of encapsulated hASCs.

An important aspect for hydrogels intended for use ascell carriers is how the encapsulation process affects thetarget cells. These hydrogels have been used to encapsu-late enzymes, proteins or drugs and then release themthrough dissolution of the hydrogel structure (Baezaet al., 2002; Spagnuolo et al., 2005; Prakash and Martoni,

Figure 3. Viability and proliferation of human adipose stem cells (hASCs) encapsulated in k-carrageenan hydrogels and cultured fordifferent time points in basal and chondrogenic medium. Fluorescent images (Calcein AM staining) showing the viability and distributionof encapsulated hASCs after 21days of culture;magnification=�5,with 100mmscale bar (A). Proliferation of hASCs cultured in basal andchondrogenic medium, results based on DNA test performed after 1, 7, 14 and 21days of culture (B). Error bars represent mean�SD. Cellnumber is statistically significantly higher at 21days than at 14days for both basal and chondrogenic conditions (p<0.05).

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2006), but the use of k-carrageenan for cell delivery is stillpoorly exploited in the literature. It seemed that a signifi-cant drop in cell proliferation from the initial time couldbe noticed and this behaviour could be due to the fact thatcells were being released from the cell-loaded hydrogelsystems during culture time, owing to degradation of thehydrogels. Gelation of k-carrageenan hydrogel wasinduced by KCl treatment, which might initially haveaffected cell viability, since for fast hydrogel formation ahigh KCl concentration was used and the remaining KClmay have influenced viability. Moreover, decreased cellviability upon cell encapsulation in hydrogels is a quitecommon finding, confirmed in several studies reportedin literature (Awad et al., 2004; Marsich et al., 2008;Wei et al., 2008).

From the DNA assay data (Figure 3B), it was possible toconfirm that until day 14 of culture the different cultureconditions induced significant changes in the proliferationof hASCs. Usually stem cell proliferation decreases whenthe differentiation process is activated; thus, the decreasein cell content could be attributed to this characteristic.The results obtained regarding the rate of proliferationof the hASCs cells during the period when they weresubmitted to chondrogenic differentiation medium indicatethat from day 14 to day 21 of culture, the cell proliferationrate increased significantly (**p< 0.05). Furthermore,hydrogels loaded with hASCs cultured in basal mediumpresented significantly lower proliferation than hASCscultured in chondrogenic differentiation medium. Simi-lar studies using other hydrogels, such as collagen, algi-nate or agarose, using either BMSCs (Bosnakovskiet al., 2006) or ASCs (Awad et al., 2004), showed similarcell behaviour. Together, these results indicate thatk-carrageenan enables the viability and proliferation ofencapsulated human ASCs. The chemical composition ofk-carrageenan hydrogels, similar to that of the nativeECM, directly affects the water content of the developedcarriers, which is expected to support the phenotype ofchondrocytes.

In this study hASCs were encapsulated, given that theyare of particular interest for ease of harvest and their po-tential in therapies to promote cartilage repair, and sincethere are numerous studies demonstrating that cells iso-lated from fat tissue can undergo chondrogenic differenti-ation when cultured in adequate in vitro conditions (Junget al., 2009; Guilak et al., 2010). Studies indicated thatbone marrow-derived MSCs exhibit better chondrogenesisthan MSCs of adipose origin, but advantages such as highavailability and high cell number makes adipose tissuea highly advantageous stem cell source to work with(Afizah et al., 2007).

3.4. H&E, alcian blue, safranin-O and toluidineblue staining

Chondrogenesis occurring in the hASCs laden hydrogelscan be proved by the detection of GAGs depositionand proteoglycans protein production (Figure 4) with

metachromatic staining for alcian blue, safranin O andtoluidine blue. Figure 4 depicts the positive staining ofhASCs encapsulated in k-carrageenan for alcian blue,safranin-O and toluidine blue staining, demonstratingstarting deposition of proteoglycans (GAGs), which iscommonly found in native articular cartilage ECM. Thestaining with alcian blue (Figure 4A–D) indicated aconcentration of acidic sulphated proteoglycans, and thecells appeared round within lacunae, which is a distinctmorphological appearance and structural characteristicspecific to cartilage. For safranin O staining, we could notethe similarity in the intensity of the colour between thenucleus and cytoplasmic substance for the initial periodof culture, and the difference in colour between thesetwo for later days in culture (Figure 4E–H). An increasein the intensity of the staining that evolved from a moreorthochromatic (blue) in the initial periods of culture toa more pronounced metachromatic (purple) staining inthe later periods was noticed for toluidine blue staining(Figure 4I–L) similarly to mast cells found in connectivetissue. This effect was more evident in the regions wherecell clusters formed in comparison to individual cells pres-ent in the hydrogels. These results show that k-carrageenanhydrogel is not only a lading system for the cell but issupporting the cells’ functionality, namely the chondrogenicdifferentiation, mimicking ECM properties observed by theinteraction and cell spreading within the hydrogel. By theend of the culture time, setting and partial chondrogenesisis taking place and a longer time in culture promises fulldifferentiation of the hASCs. k-Carrageenan hydrogelsmaintain the chondrocyte phenotype and promote carti-lage-specific ECM deposition even without growth factorstimulation, as subsequently chondrogenic featureswere detected in cell-laden hydrogel cultured in basalmedium, indicating that chondrogenic induction washydrogel-dependent.

H&E-stained sections (Figure 4M–P) show the typicaltissue morphology; cell nuclei are stained dark blue whilethe cytoplasm andECMhave varying degrees of pink staining.At the end of week 1 of culture it is possible to observe ahomogeneous distribution of cells with a smaller-sizedbasophilic nucleus (blue), while in the following weeksthe cells assumed a more rounded morphology with cyto-plasmic eosinophilic (pink) substance around the nucleus.

3.5. Immunohistochemistry and real-time qRT–PCR

To further evaluate the chondrogenic differentiation ofhASCs encapsulated in 1.5% w/v k-carrageenan hydrogels,immunohistochemical analysis of specific proteins, such astypes I and II collagen, was carried out. Figure 5A–D andE–H show that the cells were positive for collagen types IIand I, respectively, during the studied culture times, and thatthe staining intensity increased with increasing length of cul-ture, particularly for collagen type II. Differences were foundat the histostructural level between the two proteins evalu-ated, with collagen type I being detected at early culturetimes, whereas positive staining for collagen type II was

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detected for the latest culture times. However, chondrogen-esis is typically a progressive process, from a morphogeneticphase to a cytodifferentiation phase of development via anumber of precursor stages to the mature chondrocytes(Lin et al., 2006). Since a heterogeneous population wasused, it was expected to also have positive staining for colla-gen type I.With time, the cells enlarged and secreted a denseorganization around the nucleus. On close inspection, cellsfrom 21day culture were spherical in shape with cytoplasmicdepositions. Although most of the cells had retained theround shape typically expressed by chondrocytes that popu-late the deeper layers of articular cartilage (Aydelotte andKuettner, 1988), cells lying closer to the surface were flat-tened. No positive labellingwas found in the negative controlsections, given the absence of the key protein markers(Figure 5I–L). The presence of a metachromatic-stainingmatrix, the chondrocyte-like appearance of the cells andthe detection of type II collagen suggest that the tissue gener-ated by these cells is similar to native cartilage.

The chondrogenic differentiation of encapsulated hASCswas additionally evidenced by the results obtained fromreal-time qRT–PCR analysis, which allowed assessment ofthe expression, at a molecular level, of several importanttypical chondrogenic markers for hASCS encapsulated in

k-carrageenan hydrogels (Figure 6). Sustained gene expres-sion levels were registered for Sox9 (*p< 0.05) andAggrecan(#p< 0.05), increasing significantly with time from day14 until the end of the experiment. Actually, Sox9was upre-gulated to values close to 30-fold from day 1 to day 21.Collagen type II and Aggrecan are considered to be the twomajor and most important constituents of hyaline cartilageECM, since the functionality of this tissue relies mostly onthe presence of these components (Figure 6). Collagen typeII gene expression decreased fromday 7 to day 14 of culture,but was significantly increased from day 14 to day 21(**p< 0.05). The expression of Collagen type I, unlikeCollagen type II, registered a two-fold increase between days7 and 21 of culture (&p< 0.05). At the end of culture it waspossible to notice that high mRNA expression of Sox9remained, as well as gene expression of Aggrecan and ofCollagen type II. It might be mentioned that Collagen typeX, related to the hypertrophic stage of chondrogenic differ-entiation, registered a slightly increased expression overthe culture time studied. In general, from the analysis ofgene expression after 3weeks of culture, different stagesin the cell life cycle could be distinguished. Several studiesreported in the literature (Mueller et al., 2010; Zscharnacket al., 2010), investigating the chondrogenic differentiation

Figure 4. Optical microscopy images of histological sections obtained from k-carrageenan hydrogels with encapsulated hASCs, col-lected after several periods of culture and stained with alcian blue (A–D), safranin O (E–H), toluidine blue (I–L) and H&E (M–P). Scalebar=100mm; magnification=�40

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of stem cells, state that chondrogenesis starts around day14, when the cells start to express chondrogenic markers,as we also reported in this study. The time-frame of thestudy, namely 21days of culture, was selected based onprevious studies, with similar aims, using encapsulated

stem cells in hydrogel for chondrogenic differentiation(Bosnakovski et al., 2006; Hwang et al., 2006; Zhenget al., 2009). Although it would be interesting to verify theoutcomes in a long-term experiment, envisioning theclinical application of the proposed strategy, we weremostly concerned with the ability of the hydrogelsto guarantee early predifferentiation, as a long-termin vitro culture before implantation is not interesting in aclinical/industrial scenario.

Sox9 gene expression was prematurely upregulated,which indicates the early onset of chondrogenesis (Ahmedet al., 2007). Sox9 is an important regulator of the chondro-cyte phenotype (de Crombrugghe et al., 2000) and controlsthe expression of Collagen II, a well known marker of theECM of cartilage, which is usually expressed in the end-stageof the chondrogenic process of differentiation (Goldringet al., 2006). In this system, hASCs were likely stimulateddown the chondrogenic pathway by the growth factorTGFb1 present in the chondrogenic medium. These findingsindicated that hASCs can be successfully differentiated intochondrocytes when encapsulated in k-carrageenan.

3.6. Dynamic mechanical analysis (DMA)

The compressive modulus is a particularly importantparameter to consider in cartilage tissue engineering, asnative cartilage is subjected to movement (i.e. walking,running); loading and unloading are transient eventsoccurring in< 1 s. Hence, the mechanical response of car-tilage should be measured under dynamic cyclic condi-tions at a functionally relevant frequency, i.e. in the range

Figure 5. Light microscopy images obtained from immunohistochemical staining of hASCS encapsulated in k-carrageenan hydrogelsafter 21days of culture for collagen type II (A–D) and type I (E–H) and negative controls (I–L). Scale bar=100mm;magnification=�40

Figure 6. Relative expression of the chondrogenic specific genesSOX9, aggrecan, collagen X, collagen type I and collagen type II,based on the mRNA produced by encapsulated human ASCs after7, 14 and 21days of culture. The expression of these genes wasnormalized against the housekeeping gene GAPDH and calcu-lated by the ΔCT method. The symbols denote statistically signif-icant differences between day 14 and day 21 for the followinggenes: *, SOX9; **, collagen II; #, aggrecan; 8, collagen X; andthe symbol &, between day 7 and day 21 for collagen I gene.Statistical analyses were conducted using one-way ANOVA forn=3; p<0.05. Error bars represent mean�SD

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0.5–3Hz (Stolz et al., 2004; Ronken et al., 2012). Theinvestigation of the viscoelastic properties of biodegradablehydrogel systems may be of great interest, because one cannot only simulate the physiological dynamical loading butalso access relevant fundamental information at the molec-ular level, from both a structural and a dynamic perspective(Oliveira et al., 2005). Moreover, because of its complexstructure and interactions of its biochemical constituents,and due primarily to fluid flow through the solid matrix,cartilage behaves mechanically as a viscoelastic solid(Cohen et al., 1998).

DMA allowed the determination of the mechanicalproperties of k-carrageenan hydrogels with encapsulatedhASCs after different culture times (1, 7, 14 and 21days)in either basal or chondrogenic medium (as compared tok-carrageenan hydrogels without cells), while immersedin a PBS solution at 37 �C and throughout a physiologi-cally relevant range of frequencies. Storage (elastic) andloss (viscous) components of the complex modulus weredetermined and are presented in Figure 7. The storagemodulus (E0) curve shows how the stiffness of the poly-meric material changes with time in culture (Figure 7A).

Figure 7. Storage modulus (E0) obtained from dynamic mechanical analysis upon compression of plain hydrogels and hydrogels withencapsulated hASCs, cultured in basal and chondrogenic media and tested at a frequency of 1Hz (A). The frequency sweep testpresents the storage modulus in a logarithmic scale at frequencies of 0.1–10Hz of k-carrageenan hydrogels with hASCs cultured inbasal medium (B), hydrogels with hASCs exposed to chondrogenic medium (C) and hydrogels control (D). The damping factor(tan d) is displayed upon compression over a period of 21days for chondrogenic condition (E). Values reported correspond to anaverage of five test samples (n=5)�SD

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At a frequency of 1Hz, the storage modulus of the hydro-gels was estimated to be 0.23MPa for hASCs in basalmedium, 0.22MPa for cells cultured in chondrogenicmedium and 0.17MPa in k-carrageenan gels alone byday 21, revealing the elastic nature of these gels. In thisstudy, the storage modulus (E0) and phase angle or damping(tan d) were monitored as a function of frequency for thedifferent hydrogels. The storage modulus curves show howthe stiffness of the polymeric material changed withfrequency (Figure 7B–D). Increase in E0 was observedfor increasing frequencies in the range 0.1–10Hz, whichsuggests that the hydrogels exhibited stiffer behaviourfor higher frequencies. With the application of highfrequencies to the hydrogel, the material became glassyand solid-like; at very low frequencies, the polymerexhibited a more liquid-like or rubbery response. Thestorage modulus of the hydrogel without cells presentedvariations between the initial and final time points, sug-gesting that the hydrogels become softer, possibly dueto a loss of the cross-linking ions or to a loss in gel stabilitydue to thermal factors, as it is a thermoreversible andionic hydrogel. The elastic modulus of k-carrageenanhydrogels was improved with encapsulation of hASCsand with increasing time in culture (Figure 7A). Similarbehaviour was found for agarose or alginate hydrogelswith the same type of cells and similar culture conditions(Awad et al., 2004). Nevertheless, k-carrageenan withencapsulated hASCs exhibits a compression modulusapproximately 10-fold higher than that reported foragarose and alginate hydrogels in the study by Awad et al.(2004). Hydrogels also possess damping capability thatmay be useful to dissipate cyclic mechanical energy that isimposed in an implantation scenario. ’Damping’ is the termused for the general tendency of vibrating materials orstructures to lose some elastic energy to internal heatingor external friction; it tell us how good a material will beat absorbing energy (Mano et al., 2002). The correspondinggraphs are shown in Figure 7E. An increase in tan d isobserved from 0.1 to 10Hz for day 1 for the chondrogenicmedium condition, suggesting a higher dissipation capa-bility of k-carrageenan hydrogels at higher frequencies(Figure 7E). At the beginning of culture we could noticean increase in tan d values, but after 21 days in culturewe could observe a decrease for increasing frequencies,which is related to the higher viscous component ofhydrogel biomaterial.

During the 21 days in culture, DMA analysis indicatedan increase in storage modulus and in the viscoelasticproperties of k-carrageenan gels with encapsulated cells,suggesting an increase in the stiffness possible due toECM production. It is believed that the subsequent depo-sition of ECM components within the gel constructs wasfacilitated by the gradual degradation of the gels. At day21 the statistical analysis indicated that the mean differ-ence was significant at the 0.05 level between the cell-encapsulating hydrogels compared to the cell-free hydro-gel control. The mechanism behind the increase in stiffnessof the cell-laden hydrogels likely involves more than thecell load, as it is a result of cell-mediated interactions

and deposition of matrix with time. In addition, stemcells can proliferate as well as differentiate, but as cellsdifferentiate their rate of replication usually decreases,and by the end of 21 days there is no difference interms of elastic modulus values between cells exposedor not exposed to the differentiation medium. It hasbeen demonstrated that the biomechanical propertiesof articular cartilage greatly depend on composition,density of ECM and interstitial fluid flow (water andsolutes) (Evans and Quinn, 2005). Cell encapsulationand ECM deposition may therefore result in progressiveincrease of the mechanical properties of the 3D struc-tures, as shown previously for the cell–hydrogel con-struct (Miyata et al., 2004).

The characterization at the molecular level correlatedwith the mechanical properties developed by thesesystems, as at day 14 we observed a decrease in theCollagen type II chondrogenic marker (responsible forthe tensile properties); at the same time point there isan upregulation of Aggrecan gene expression (responsi-ble for the compressive properties of the hydrogel), asthese markers are interacting and are being expresseddifferently during chondrogenic differentiation. Thesevalues mimic the mechanical properties found in articu-lar cartilage and they are higher or within the range ofvalues found for other hydrogels used in similar carti-lage-regenerative approaches (Kisiday et al., 2002; Nettleset al., 2004). Human articular cartilage possesses signifi-cant mechanical properties, with a compressive modulusof 0.79MPa, a shear modulus of 0.69MPa and a tensilemodulus of 0.3–10.2MPa (Fisher et al., 2004). Evaluationof these properties contributed to a further understandingof the potential of these hydrogels for the targetapplication.

Considering the suitable mechanical properties,together with the simplicity and reproducibility of thepreparation process for k-carrageenan hydrogel forma-tion, which is performed under mild conditions withoutemploying any extraneous toxic crosslinking agents, it ispossible to conclude that such a matrix has potentialapplications in cartilage tissue-engineering applications.To further enhance the quality of engineered cartilagetissues formed from encapsulating hASCs within thissystem, one may optimize the release rates of supplemen-tal growth factors incorporated in the hydrogels. It is alsopossible that host cells could be recruited to the gel, byrelease of the growth factors participating in cartilageformation.

4. Conclusions

This study presents k-carrageenan as a potentialhydrogel for cell delivery, with application in the regener-ation of cartilage. In summary, the results obtained usingk-carrageenan hydrogels as a cell-loading matrix, pre-pared via an ionic crosslinking reaction, demonstratethat this system could be an alternative cell-deliveryhydrogel system. hASCs encapsulated in k-carrageenan

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hydrogels remain viable, proliferate and differentiate intothe chondrogenic lineage. Furthermore, k-carrageenanhydrogels with encapsulated hASCs were able toachieve mechanical properties, under compression, inthe range of those reported for native cartilage. Thesepromising in vitro results should be further analysed inanimal model experiments designed to address funda-mental questions concerning the in vivo degradationof the hydrogel and the possible in vivo differentiation ofencapsulated cells.

Conflict of interest

The authors have declared that there is no conflict of interest.

Acknowledgements

Elena G. Popa would like to acknowledge the PortugueseFoundation for Science and Technology (PhD Grant No. SFRH/BD/64070/2009).

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