Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies

Soft Matter

PAPER

a3B’s Research Group – Biomaterials, Biode

Minho, Headquarters of the European Insti

and Regenerative Medicine, AvePark, 4806-9

[email protected]; Fax: +351 253 5bICVS/3B’s – PT Government Associate Labo

Cite this: Soft Matter, 2013, 9, 875

Received 9th August 2012Accepted 25th October 2012

DOI: 10.1039/c2sm26846a

www.rsc.org/softmatter

This journal is ª The Royal Society of

Cryopreservation of cell laden natural origin hydrogelsfor cartilage regeneration strategies

Elena G. Popa,ab Marcia T. Rodrigues,ab Daniela F. Coutinho,ab Mariana B. Oliveira,ab

Jo~ao F. Mano,ab Rui L. Reisab and Manuela E. Gomes*ab

The time span needed for obtaining a functional cartilage substitute using tissue engineering strategies,

together with the need for specific patient oriented constructs has stimulated the growing interest for

developing “off-the shelf” products. One way to deliver such products is based on long-term storage

processes, such as cryopreservation, that will provide the clinical substitute available as needed and

could be adapted to an autologous immediate solution for the patient. The aim of this study was to

examine the effects of cryopreservation on the chondrogenic differentiation characteristics of human

mesenchymal derived stem cells isolated from adipose tissue and encapsulated in k-carrageenan

hydrogels. These bioengineered constructs are anticipated to participate in a cartilage regeneration

strategy providing temporary habitation for cell survival, proliferation and production of an extracellular

matrix which is expected to replace the hydrogel, enhancing the regeneration of native tissues in clinical

settings. The results obtained show that the hydrogels withstand the cryopreservation with dimethyl

sulfoxide, maintaining their structural integrity, while assisting cells proliferation and chondrogenic

potential after cryopreservation. Thus, cell encapsulation systems of natural based hydrogels seem to be

an interesting approach for the preservation of cartilage tissue engineered products.

Introduction

One of the main prospects of cartilage tissue engineering is thepossibility of developing custom-made solutions for regenera-tive medicine on an individual patient basis, a major challengebeing the preservation and storage of the bioengineeredconstructs aimed at ready-to-use applications. Since the processof developing tissue engineered constructs is typically timedemanding, approaches to facilitate and accelerate their clin-ical use have emerged. Assuming this, the cryopreservation ofcells, as well as more complex systems such as cell encapsula-tion devices, tissue-engineered constructs, and even laboratory-produced tissues and organs may be critical in the future. Thus,the scientic increment in the literature and current clinicalexperience for the repair of injured or diseased articular carti-lage supports the need for providing such novel products likecultured cells on engineered tissue constructs to improve theclinical market options. Cryopreservation of various mamma-lian cell types has been a standard procedure for many yearsenvisioning cell preservation for future outcomes.1–3 Cellencapsulation is one of the primary techniques to colonizetissue-engineered hydrogels as cells are immunoprotected from

gradables and Biomimetics, University of

tute of Excellence on Tissue Engineering

09 Taipas, Guimar~aes, Portugal. E-mail:

10 909; Tel: +351 253 510 906

ratory, Braga/Guimar~aes, Portugal

Chemistry 2013

the host environment once implanted. The most applied cryo-preservation protocols use dimethyl sulfoxide (DMSO) (1 to 20%v/v) in the serum-supplemented culture medium combinedwith slow-freezing rates.4 However, the cryopreservation oftissue engineered constructs is typically associated with diffi-culties in preserving the 3D structural properties of theconstructs, and the challenge stands for reducing to minimumthe cryo-injury, predominantly related to ice formation uponfreezing and thawing.5 Nevertheless, complex cell-scaffoldconstructs kept under routine cryopreservation procedures areemerging as potential banking constructs in regenerativemedicine applications.6,7 Alternatives have been proposed withthe use of several cryoprotectants,8,9 vitrication solutions,10,11

or the entrapment of cells on an articial extracellular matrix(ECM), like hydrogels.12,13 Articial ECM/hydrogels may beviable to protect the cells from outside damage, whereas thecryoprotectants will protect the cell from intracellular or inter-cellular ice crystals formation. This system allows cells to beprotected against mechanical damages during ice crystalliza-tion and the danger of disrupting cell–cell interactions isreduced through immobilization.14,15 Natural hydrogels havebeen widely used in the biomedical eld, as cell encapsulationand delivery systems, envisioning their application in severalregenerative medicine therapies.16–18 Among the different poly-mers studied, k-carrageenan is a very versatile, thermosensitivehydrogel used to develop ionotropic matrices for in situ immo-bilization of cells.19 Carrageenan belongs to a family of linear,

Soft Matter, 2013, 9, 875–885 | 875

Fig. 2 Optical microscopy images of hASCs–hydrogel construct and kCR

Soft Matter Paper

water soluble, sulfated, anionic polysaccharides extracted frommarine red algae.20,21 They are highly exible molecules with theability to shape into different formats at room temperature dueto its thermosensitive characteristic. The formation of irre-versible carrageenan hydrogels can be obtained in the presenceof potassium ions, which leads to the formation of more stablehydrogels. More recently, k-carrageenan based systems havebeen proposed for cartilage regeneration strategies22,23 and fordelivery systems of proteins and growth factors by ourgroup.24–26 Although the cryopreservation of cells or tissues is acommon method that provides advantages for cellular thera-pies,27 few studies have addressed the impact of cryopreservingtissue bioengineered constructs as a combined cell therapyapproach.28 A fundamental objective of this work was to verifythe viability, proliferative and differentiation capacity of humanadipose derived stem cells (hASCs) into chondrocyte-like cellswhile encapsulated in the k-carrageenan hydrogels (kCR),demonstrating the ability to maintain the metabolic function-ality aer cryopreservation. Another crucial goal towards theapplicability of the proposed system into clinical settings wasthe analysis of the effect of cryopreservation on the morpho-logical and mechanical properties of the system as a functionalconstruct. The outcomes of this study suggest that this strategycould provide a ready-to-use pool of cells for cartilage clinicaltreatments.

hydrogel without cells collected before (BC) and after (AC) cryopreservation. Theinsets represent the full image of the discs, whose details are shown in themagnified image. Scale bars represent 100 mm.
Results
Cell laden hydrogel morphology before and aer thecryopreservation process

In the present study we have analyzed the effect of the cryo-preservation process on the chondrogenic features developed bythe hASCs aer encapsulation in kCR sulfated hydrogels. Cellladen hydrogels were cultured for 2 weeks before being cryo-preserved for one month under standard conditions asdescribed in the Experimental section, the cryopreservationprocess. Aerwards, constructs were thawed and cultured for anadditional 7 days period (Fig. 1).

In order to analyze the morphology of hydrogels and theencapsulated cells, micrograph images were obtained from thehASCs–hydrogel constructs culture in chondrogenic medium(CM) and basal medium (BM), before and aer cryopreservation

Fig. 1 Study design showing details of the preparation and the cryopreservationprocess for the developed hASCs–hydrogel construct.

876 | Soft Matter, 2013, 9, 875–885

(Fig. 2). Macroscopically, the integrity of the hydrogels did notseem to be affected by the cryopreservation–thawing process,considering the maintenance of the shape aer cryopreserva-tion, with minor variations in the hydrogel volume (Fig. 2insets). Exceptions in terms of surface morphology were foundin the control hydrogel (without cells) where it is possible toobserve shrinkage of the cryopreserved sample and wrinkles onthe surface. In cell laden hydrogels, a better spreading of thehASCs before cryopreservation compared to aer cryopreserva-tion is noticeable, when cell appeared more rounded with lessvisible cell to cell connections. Even so, microscopic imagessuggest that the cellular density is maintained aer cryopres-ervation and that the hydrogel stability is overall maintained,keeping its structural cylindrical shape and the cells inside thehydrogels. Nevertheless, kCR hydrogels are sensitive to ionicenvironments, naturally present in cell culture media. Thus,despite kCR hydrogels showing a perfect shape immediatelyaer cryopreservation, the materials seem to be less stable aer7 days in culture, yet without compromising their functionalityas a cellular laden system.

Mechanical properties

A preliminary assessment of the stability of these hydrogelsupon cryopreservation was performed by dynamic mechanicalanalysis (DMA). DMA allowed determination of the mechanicalproperties of kCR hydrogels with encapsulated hASCs aer

This journal is ª The Royal Society of Chemistry 2013

Table 1 Summary of the conditions and total number of cell–hydrogel construct samples analysed in each characterization (retrieved from 4 independent experi-ments performed in the study). Constructs were cultured in either the basal medium (BM) or the chondrogenic medium (CM). Characterization assays were performedon samples collected before (BC) and after cryopreservation (AC)a

Groups

Experimentalcondition

Lightmicroscopy DMA

Live–deadassay dsDNA

Histology*

RT-PCRBC AC TB H&E Coll II Coll I

Cell–hydrogelconstruct tested

CM CM 3 20 3 24 12 12 12 12 18BM BM 3 20 3 24 12 12 12 12 18kCR kCR 3 20 nd 24 nd nd nd nd —

a The symbol * relates to the number of slides obtained from cell–hydrogel constructs. CM: chondrogenic medium; BM: basic medium; kCR: kappa-carrageenan hydrogel; BC: before cryopreservation; AC: aer cryopreservation; – data not showed; DMA: dynamic mechanical analysis; nd: notdetermined; dsDNA: double strand DNA; TB: Toluidine Blue; H&E: haematoxylin and eosin; Coll II: collagen type II; Coll I: collagen type I andRT-PCR: reverse transcription polymerase chain reaction.

Paper Soft Matter

different culturing times (14 days before cryopreservation and 7days aer cryopreservation) in either the chondrogenic or basalmedium (as compared to kCR hydrogels without cells). Moredetails on the testing conditions were explained in Table 1 andin the Characterization section. The analysis was performedwith the hydrogels immersed in a KCl solution at 37 �C andthroughout a physiological relevant range of frequencies.Storage (elastic) and loss (viscous) components of the complexmodulus were determined and are presented in Fig. 3. Theelastic modulus (E0) curve shows the viscoelastic behavior ofkCR hydrogels with or without cells before and aer cryopres-ervation and how the stiffness of the polymeric materialchanges with the frequency. Increase in E0 is observed for theconditions which were not exposed to the freeze–thaw cycles ascompared to the samples that were cryopreserved and are notfrequencies dependent. The storage modulus for the hASCs–hydrogel exposed to the chondrogenic medium before cryo-preservation holds the highest values, showing a stiffer struc-ture, and tends to increase with increasing frequency from 85 to120 kPa (Fig. 3). The inuence on the loss factor (tan d) of thefreezing process and the growth factor addition to the mediumis also presented in Fig. 3. All hydrogels showed a frequency-dependent behavior, with damping increase in frequencieshigher than 2 Hz. In general, a more prominent increase wasobserved in the hydrogels exposed to the freeze–thaw cycles.

Fig. 3 (a) Storage modulus (E0) and (b) loss angle (tan d) obtained from dynamicmechanical analysis upon compression of hydrogels with encapsulated hASCsand plain hydrogels cultured in chondrogenic and basal media before and aftercryopreservation.

Viability and proliferation of hASCs encapsulated in k-carrageenan hydrogels

Fluorescence microscopy images provided information aboutcell viability and morphology and the cell content wasevaluated by DNA quantication. Fig. 4a depicts the live/dead imaging of hASCs encapsulated in kCR hydrogels andcultured in the chondrogenic or basal medium. Stained ingreen are the viable cells, which enzymatically converted thenon-uorescent cell-permeant calcein AM into the intenselyuorescent calcein. A higher hASCs density in the hydrogelconstructs is observed prior to cryopreservation with a lowernumber of positive viable cells present aer cryopreserva-tion. In the chondrogenic formulation, cells appear to bemore agglomerated and spread compared to the basal


conditions for the earlier time point. Aer cryopreservation,cells cultured in the chondrogenic medium seem to be morebiologically active than the ones cultured in basalconditions.

The DNA content corroborated with the live/dead dataanalysis. Cell content measurements (Fig. 4b–d) tend todecrease aer cryopreservation. A decrease in the cell contentwas registered (p < 0.05) aer cryopreservation both forconstructs cultured in basal or chondrogenic media, but higher

Soft Matter, 2013, 9, 875–885 | 877

Soft Matter Paper

values (p < 0.05) were registered for the samples cultured in thechondrogenic medium both BC and AC (Fig. 4b). The cellularcontent of the hASCs–hydrogel construct and the pellet culturedin the chondrogenic medium evidenced a similar pattern, whencompared to the respective samples cultured in the basalmedium (Fig. 4c and d). Nevertheless, the cellular contentdecrease is less accentuated in hASCs–hydrogel constructs thanin the pellets, suggesting that the hydrogels supported cellcontent maintenance in culture post-thawing.

Histological analysis

Histological evaluation provided qualitative information todetect possible changes in cell morphology, ECM production,and the overall hydrogel structure before and aer cryopreser-vation. Fig. 5 shows the light microscopy images ofthe constructs stained with toluidine blue (TB) and

Fig. 4 Cell viability (a) and proliferation (b)–(d) of hASCs–hydrogel constructs cucryopreservation (AC). (a) Merged green/red fluorescence images of hASCs laden h(red). Scale bars represent 100 mm. (b) Cell content obtained from dsDNA quantificastandard deviation. Statistical significant differences in the cell amount for the chonproliferation tendency based on dsDNA quantification for the constructs and the peproliferation under the same conditions as the hASCs–hydrogel construct. D1 correervation, D0 stands for immediate thawing and D7 refers to 7 days in culture afterbetween D14 and D0.

878 | Soft Matter, 2013, 9, 875–885

hematoxylin-eosin (H&E) stains. H&E showed well distributedhASCs throughout the hydrogel section in chondrogenic orbasal media either before or aer cryopreservation. Also, thehigh amount of hASCs observed indicated that cells weresuccessfully encapsulated in the hydrogels and remainedenclosed in the polymeric structure during the cryopreserva-tion–thawing cycle, not being particularly affected by the minorhydrogel ssures caused by cryopreservation (Fig. 5). TB stain-ing revealed the presence of negatively charged glycosamino-glycans (GAGs) in the cross-sections of the hASC–hydrogelconstructs (Fig. 5). The localization of GAGs, one of the majorcartilage ECM components, indicates possible deposition of acartilage-like ECM. ECM localization is detected before andaer cryopreservation indicating that the freeze–thaw protocoldidn’t affect extensively the GAGs production, and consequentlythe intrinsic metabolism of differentiated hASCs. Aer cryo-preservation and in chondrogenic conditions, hASCs tend to

ltured in either the chondrogenic or the basal medium, before (BC) and afterydrogels, calcein-AM indicates live cells (green) and propidium iodide dead cellstion assay for chondrogenic (CM) and basal (BM) media. Values indicate mean �drogenic or basal medium BC and AC were registered (p < 0.05). (c) and (d) Cellllet control group. The pellets were used as an experimental control to assess cellsponds to one day in culture, D14 represents 2 weeks in culture before cryopres-cryopreservation. The cryopreservation period is indicated by the dashed lines in


Paper Soft Matter

agglomerate into multiple-cell lacunae. The micrographs alsoevidence that cells are individually or paired gathered insidevacuole-like structures typically related to the chondrocytesmorphology and to native cartilage features. When encapsu-lated in kCR hydrogels, hASCs adopted a more sphericalmorphology, which is typically observed in encapsulationsystems.

Immunohistochemisty

Complementarily, immunohistochemical analysis of the bio-macromolecules typically present during cartilage developmentwas performed. Distribution of the cartilage-like matrix wasevaluated through immunolocalization of cartilage matrixproteins before and aer the freeze–thaw process and theposterior culture for additional 7 days in the chondrogenic orbasal medium. Before cryopreservation, the deposition ofcollagen type II seems to be higher in the chondrogenicformulation when compared to collagen type I. Fig. 6 shows thatcells present in the kCR hydrogel were positively stained forboth types of collagens and these cartilage matrix componentswere homogeneously distributed throughout the hydrogels.Before cryopreservation, the hASCs cultured in the basalmedium seem to show lower expression of collagen II andcollagen I when compared to the chondrogenic samples. Aercryopreservation, the expression of collagen I seems to decreasein hASCs–hydrogels cultured in the basal medium, evidenced bya decrease in the brownish color revealed by DAB, and increasedblue associated with hematoxylin counterstain. Moreover, thepresence of cells in lacunae, characteristic of native articularcartilage tissue was highlighted. Overall, the cells maintainedtheir chondrogenic properties throughout the culturing timebefore the freeze–thaw cycle. The intensity of collagen type Iimmunolocalization seems to increase in the chondrogeniccondition aer cryopreservation. Furthermore, collagen type IIand collagen type I expressions appear to be similar in the basalmedium aer cryopreservation. These cartilage matrix markerswere distributed throughout the hASCs–hydrogel construct.

Fig. 5 Hematoxylin–eosin (H&E) staining for cellular detection and overall cell distribnamely glycosaminoglycans (by Toluidine Blue, TB) of chondrogenically induced hhydrogel constructs were collected and stained BC and AC. Scale bars represent 10


Colony formation in the medium supplemented with TGF-b1,aer cryopreservation is detected, as previously referred. Overalland despite minor variations, cryopreservation seems not tointerfere in the expression of proteins associated with cartilageECM.

RT-PCR

To complement the immunostaining results, RT-PCR was per-formed in hASCs–hydrogel BC and AC. The results obtainedfrom the genotypic evaluation of cartilage-related genesexpressed by hASCs within kCR hydrogels are presented inFig. 7. The expression of the cartilage specic genes was veriedin an agarose gel, using GAPDH as housekeeping gene. Theexpression of transcripts associated with cartilage (i.e. SOX9,aggrecan, collagen type I, collagen type II and collagen type X)was observed by 14 days of culture, just before cryopreservation.hASCs encapsulated in kCR showed high levels of collagen II,collagen X, and SOX9 in chondrogenic conditions. Conversely,aggrecan seems to be more expressed in the basal medium,while collagen I is similarly expressed in the basic or chondro-genic medium. In general, aer cryopreservation, gene expres-sion seems to be lower, almost undetected, in all conditionsstudied, except for the housekeeping gene and collagen X.

Discussion

Presently, cryopreservation protocols are the only means forlong term preservation of biological samples, thus playing animportant role in cell and tissue banking assuming a greaterimportance when tissue engineering is approaching aneveryday reality.29 The relevance in exploiting “ready-to-use”cartilage engineered constructs fullling patient-specic char-acteristics combined with adequate preservation methodolo-gies, anticipates the need for development of “out of shelf”tissue substitutes. It is envisioned that such approaches willplay a central role in future applications with tissue engineeredproducts, as constructs could be prepared in advance, and

ution within the hydrogel. Detection of cartilage extracellular matrix components,ASCs and undifferentiated hASCs encapsulated in kCR hydrogel discs. hASCS–0 mm.

Soft Matter, 2013, 9, 875–885 | 879

Fig. 6 Immunohistochemical localization of collagen type II and collagen type I proteins in hASCs–hydrogels exposed to the chondrogenic or basal medium beforeand after cryopreservation. Scale bar ¼ 100 mm.

Fig. 7 Representative image of the electrophoresis gels reporting the expressionof cartilage-related genes by RT-PCR. The gel shows the results obtained beforeand after the cryopreservation protocol and cultured in the chondrogenic or basalmedium.

Soft Matter Paper

scaled up assuring immediate availability and accessibility forclinical use.

This study investigates the inuence of cryopreservationusing a standard protocol in a promising engineered productcomposed of hASCs encapsulated in kCR hydrogels. Moreover,we proposed to evaluate if the chondrogenic potential as well ascellular viability and proliferative capacity of encapsulatedhuman adipose derived stem cells is restored aer establishedfreeze–thaw cycles. The method selected for cryopreservation ofthe developed system combines the cryoprotectant DMSO withFBS, using temperature gradient cycles before a long termpreservation in liquid nitrogen (�196 �C), followed by a quickthawing procedure. Cooling temperature cycles wereapproached considering that gradual freezing reduces the riskof ice crystal formation and cell damage. At a macroscopic level,evidence suggested that this procedure does not signicantlyaffect hydrogel structural integrity, as the morphology and

880 | Soft Matter, 2013, 9, 875–885

stability of hASCs–kCR hydrogels were not altered radically bythe cryopreservation process. The ssures observed on thesurface of the hydrogel are not interfering with the overallconstitution of the hydrogel (Fig. 2). The fact that k-carrageenanhydrogels successfully underwent cryopreservation whilepreserving their structural integrity indicates that these carriersare easily manipulated and suitable to be used as efficientencapsulation systems under simple cryopreservation proto-cols. This statement is in accordance with literature reportswhich evidence the good freeze–thaw stability of carrageenanbased hydrogels.30

Moreover, dynamic mechanical analysis (DMA) was per-formed to better characterize the mechanical/viscoelastic prop-erties of the hydrogels in a simulated physiological environment(wet state at 37 �C). The non-cryopreserved constructs, registeredan increase with frequency which is not detected for the cry-opreserved construct. During the days in culture prior to cryo-preservation, DMA analysis indicated an increase in storagemodulus and in viscoelastic properties of kCR gels with encap-sulated cells suggesting an increase in the stiffness, possibly dueto the extracellularmatrix production. Fig. 3 shows that in all thefrequency range, the storage modulus is high for the hASCs–hydrogel construct chondrogenically committed, which corre-sponds to the typical stiffness effect foundmore in these systemsdue to the cellular component and high secretion of ECM. DMAdata indicated that the hASCs–hydrogels show a viscoelasticbehavior and maintain their mechanical properties aer afreeze–thaw process. Furthermore, obtained values weredescribed to be in the range of constructs aimed at cartilageengineering applicationswith a compressionmodulus similar tothe ones reported for agarose and alginate hydrogels.31 Thevalues of tan d (higher than 0.2) reveal a clear viscoelasticbehavior of the hydrogels. This is an important feature inbiomaterials used for tissues subjected to periodic loads, such ascartilage, since it shows the material’s ability toabsorbmechanical energy. The increase of the loss factor for thefreeze–thawed gels, alongwith the stability observed for E0 values


Paper Soft Matter

with frequency shows that for high frequencies these hydrogelsincrease their ability to dissipate energy. Nonetheless, atfrequencies corresponding to human movements, such aswalking or running (around 1 Hz) no signicant differences inthe loss factor of the BC and AC hydrogels were observed.

Previous reports about the functional application of kCRhydrogels show their applicability for in situ encapsulation ofdifferent types of cells, while keeping cellular proliferation andassisting the expressionof cartilageextracellularmatrixmarkers.19

Microscopy analysis of hASC–kCR hydrogels revealed low cellulardensity andminormorphological variations in cell shape and cell-to-cell interactions aer cryopreservation. In the particular case ofencapsulated cells cultured in BM, morphological changes aremore signicant AC as they lose most of pseudopodia and theirfusiform appearance into a more chondrocytic shape. Thismodicationmaybe associatedwithcell adjustment to the freeze–thaw cycle, which is more evident in a basic culture mediumwithout chondrogenic biochemical supplements.

Although the ice nucleation was not controlled by freezingsystem equipment, the damage of ice formation/nucleationaffecting the hASCs encapsulated in kCR hydrogels was indi-rectly assessed by live/dead and dsDNA quantication tests.Overall, and despite an expected decrease observed upon cryo-preservation, the constructs were able to maintain the cellsviability and proliferation, suggesting the intra- and extra-cellular formation of ice crystals was minimal. It is known thatthe freezing–thawing process interferes with the biophysicaland biochemical features of the cells, resulting in alterations oftheir functional competency and survival.32 As a consequence,cryopreserved cells require a recovery time aer thawing foradjusting sudden and radical micro-environmental changes.The observed decrease in the cell content post-cryopreservationmight also be explained by the minor ssures observed in thekCR hydrogels post-cryopreservation (Fig. 2), which could led tothe release of some encapsulated cells from the system.

Immediately aer thawing, hASCs viability/cell content levelswere higher compared to longer time in culture and tend todecrease with time. This trend was found both for hASCs–kCRconstructs and pellets. The explanation may reside in the factthat short time in culture aer cryopreservation is not sufficientto allow cellular reboot. While the rationale behind the shorttime in culture aer cryopreservation was to study the imme-diate availability and functionality of the cell–hydrogelconstruct, longer time in culture is expected to assure thecellular recovery from the cryopreservation process as reportedpreviously.33

The more accentuated decrease in the cell content observedin the pellets, as compared to the cell-laden hydrogels, suggeststhat hydrogels may act like a protective barrier to intracellularice formation or as a buffer for the cryoprotectant agent diffu-sion, as it has been previously reported,14 yet allowing theexchange of nutrients and oxygen.

Interestingly, the cell contents registered for hydrogelscultured in chondrogenic conditions are signicantly higher(p < 0.05) than in basal medium cultures, before and aercryopreservation. The combination of kCR hydrogel and chon-drogenic supplements may provide the necessary stimuli for


cellular colonization despite chondrogenic differentiation.Histological ndings indicated cellular morphology changesaer the cryopreservation process. ECM proteoglycans deposi-tion was observed in the pericellular regions of most cell clus-ters. Although adult chondrocytes do not divide or establishcell-to-cell contacts, these cells are responsible to produce acartilagineous dense ECM,34 thus maintaining cartilage integ-rity and function. This nding may be explanatory to the factthat stem cell chondrogenically differentiated produced andsecreted ECM components. The pre-existing lacunae observedin the histological micrographs (Fig. 5) formed before thecryopreservation cycles, typical to the chondrocytes phenotype,enlarge aer cryopreservation and were colonized withmigrating cells, creating cell agglomeration into the free spaces.This nding may be associated with the fact that enhancedtissue deposition is observed nearby these aggregate structures.H&E control staining revealed cells entrapped in the hydrogelsand a good stability of the complex cell–hydrogel system.

The immunolocalization of the proteins analyzed in thisstudy (collagen type II and I) is known to be naturally present incartilage ECM. Collagen type II is typically detected in elasticcartilage while collagen type I is involved in bril formation ofseveral skeletal tissues and expressed in brocartilage, a scartissue, in vivo. Both forms of collagen II and collagen I wereproduced and retained in the form of ECM to an analogousextent before and aer cryopreservation process (Fig. 6).Moreover, a positive localization was detected for the cellscultured in the basal medium, i.e., in the absence of anychondrogenic inducer growth factor, suggesting that the 3Dhydrogel environment was sufficient to stimulate the adiposederived stem cells to present typical cartilage-like morphology,namely vacuoles and rounded shape, as well as the synthesis ofthe collagen II rich matrix. The deposition of the extracellularmatrix resulted from the 2 weeks of cell culturing prior tocryopreservation. This time point was selected based onprevious studies on hASCs chondrogenic differentiation.19 Two-weeks were the shortest culturing time necessary to promotechondrogenic differentiation of hASCs with ECM production.The ECM observed in both chondrogenic and basal culturesmay inuence the cryopreservation effect on the hASCs–kCRconstructs. Similarly to the functionality of the kCR hydrogels,the deposition of ECM may allow a controlled diffusion of thecryoprotectant into the cells located in the matrix.35

Genes commonly studied for chondrogenic differentiationanalysis include collagen types I, II and X, as well as SOX9 andaggrecan. SOX9, a marker of early chondrogenesis, is alsoimportant in achieving a healthy cartilage mainly because SOX9stimulates aggrecan and collagen II synthesis.36 AlthoughCollagen X has been associated with hypertrophic chondrocytesin vivo, it has also been described as a lineage specicmarker forcartilage differentiation of mesenchymal stem cells.37 It is wellestablished that multipotent mesenchymal stromal cells (MSCs)undergoing chondrogenesis have a tendency to express colla-gens I and X in vitro.38 Overall, the genes associated with thechondrogenic differentiation process are increased in hASCs–hydrogel constructs before cryopreservation for both cultureconditions (CM and BM). Aer cryopreservation, gene

Soft Matter, 2013, 9, 875–885 | 881

Soft Matter Paper

expressions seem to be reduced under all conditions except forthe housekeeping gene and for collagen X in the chondrogenicmedium. The decrease in gene expression aer cryopreservationhas been described in the literature39 andmay be associatedwiththe fact that cells are still recovering from the cryopreservationprocess and are still adapting to the new conditions. Althoughsome cellular responses were already restored 7 days aer cryo-preservation, it is expected that later in time, cells will fullyrecover and express the chondrogenic related genes into thelevels prior to cryopreservation.40 Nevertheless, and since we areenvisioning an “off-the-shelf” bioengineered product withimmediate availability and ready-to-use application, a timecompromisemust be reached.That is, aer cryopreservation, thecell-kCRsystemmust be cultured tobecome functional. The timein culture is required to sustain the system functionality, thusfullling the aim it was developed for.

The broad range of biological properties of MSCs attractedthe interest of many researchers to be used for diverse thera-peutic applications. One advantage of using MSCs in thissystem rely on the fact that these cells possess a high plasticityand are dynamic in terms of cellular metabolic machinery,41

reacting more promptly to physicochemical variations in theenvironment than primary chondrocytes, which tend to dedif-ferentiate when cultured in vitro.42 Furthermore, the applica-bility of stem cells in cryopreservation systems can disclose aneven more promising approach of these cryopreserved systemsinto the regenerative medicine eld, as stem cells are immuneprivileged and these products may t different patients.43

ExperimentalPreparation of ionic k-carrageenan hydrogels

The ionic gelation process of the natural polysaccharide basedk-carrageenan (kCR, 22048, Sigma) was performed as describedelsewhere.19 Briey, the kCR powder was mixed with distilledwater to obtain the 1.5% (w/v) polymeric concentration. Thesolution was warmed up in a water bath at 50 �C, stirring untilthe complete dissolution and aerwards sterilized (121 �C for30 min) before cell culture studies. In order to form hydrogelsamples in the form of discs, 5 mL of kCR solution was pouredinto sterile plastic Petri dishes of 55 mm B and the freshlyformed hydrogels were punched out into a disc shaped sampleof 5 mmB � 3 mm height. The hydrogel formation was furtherstabilized by cross-linking with 5% (w/v) potassium chloride(KCl, P5405, Sigma) for 15 minutes. Aerwards the hydrogelsamples were washed with PBS to remove the excess of KClpresent on the surface. The hydrogel preparation is schemati-cally depicted in Fig. 1.

hASCs isolation and expansion

Human subcutaneous fat tissue samples were obtained underprotocols established with the Plastic Surgery Department ofHospital da Prelada (Porto, Portugal). Adipose tissue derivedstem cells from four female donors with a mean age of 35.25years (�8.55) and BMI of 26.26 (�2.74) were isolated by enzy-matic digestion as previously described.44 Briey, the adipose

882 | Soft Matter, 2013, 9, 875–885

tissue samples were digested with 0.075% collagenase type II(C6885, Sigma) in phosphate buffer saline (DPBS, 21600-044,Invitrogen) for 45 min at 37 �C under gentle stirring. Thedigested tissue was ltered with a 100 mm lter mesh, andcentrifuged at 1000g for 10 min at 5 �C. Aerwards the cellsuspension was washed for 10 min with lysis buffer (155 mMNH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH ¼ 7.3) to remove theerythrocytes, and additionally centrifuged at 800g for 10 min at5 �C. The adherent hASCs were expanded in the basal mediumcomposed of Minimum Essential Medium alpha (MEM, 12000-063, Invitrogen) with 10% (v/v) fetal bovine serum (FBS, 10270,Invitrogen), 1% (v/v) antibiotic–antimycotic solution (15240-062, Gibco�), and 2.2 mg mL�1 sodium bicarbonate (NaHCO3,S5761, Sigma) with the medium changed every three to fourdays. Cells were subcultured at a cell density of 3.5 � 103 cellscm�2 until achieving a sufficient cell number to be used in theexperimental assay. The hASCs used in this study were atpassage three.

Cells encapsulation

The kCR aqueous solution and the cell suspension were mixeduntil complete homogenization, and hydrogel disc samplesloaded with hASCs were formed as described above, usingsterile moulds. The hASCs were encapsulated at a density of 5�106 cells cm�3 into the kCR hydrogels and cultured for 14 dayseither in the chondrogenic or basal medium. The chondrogenicinduction medium was composed of Dulbecco’s ModiedEagle’s Medium-low glucose (DMEM, D5523, Sigma), supple-mented with 3.8 mg mL�1 NAHCO3, 1% (v/v) antibiotic–anti-mycotic solution, 10% (v/v) FBS (fetal bovine serum,SH3007103, Fisher Scientic), 1 � ITS + 1 (insulin–transferrin–selenium – liquid media supplement, I2521, Sigma), 17 mML-ascorbic acid (A4544, Sigma), 0.1 M sodium pyruvate (P4562,Sigma), 35 mM L-proline (P5607, Sigma), 1 mM dexamethasone(D2915, Sigma) and 10 ng mL�1 of human transforming growthfactor-b1 (TGF-b1, 14-8348, eBioscience). kCR hydrogel sampleswithout cells kept in culture for the same time points under thesame cell maintenance conditions and cell pellets, were used ascontrols. For the preparation of the 3D micromass pellets,aliquots of 2 � 105 cells in 0.3 mL of the medium were centri-fuged at 1500 rpm for 15 min. Pellets were cultured for 14 daysin a 37 �C humidied atmosphere with 5% CO2, and themedium was changed once a week. At the end of cell culturing,part of the samples was retrieved, rinsed in PBS solution beforeproceeding to characterization analysis as detailed in Table 1.Simultaneously, the remaining part of the samples was removedfrom culture and cryopreserved (Fig. 1).

Cryopreservation process

Samples that underwent cryopreservation were frozen for onemonth before being cultured for additional 7 days in BM or CM.The cryopreservation process was based on the followingprotocol: each sample was individually placed in a poly-propylene cryovial (479-0821, VWR) containing a solution of10% (v/v) dimethyl sulphoxide (CryoSure DMSO, 11-32-30216,Wak-Chemie Medical GMBH) in FBS. The samples were cooled


Paper Soft Matter

down to 4 �C using an ice bath (30 minutes). Aerwards, cryo-vials were moved into a conventional freezer (�20 �C; 1–2 h) andthen moved into a �80 �C freezer. Aer 12 hours at �80 �C,samples were stored inside a liquid nitrogen tank (StatebourneBiosystem) at �196 �C for one month (Fig. 1).

In total, the experiment set-up included three groups: (i)hASCs–kCR hydrogels construct cultured in the chondrogenicmedium (CM); (ii) hASCs–kCR hydrogels construct cultured inthe basal medium (BM) and (iii) kCR hydrogels cultured in thebasal medium (Table 1). The pellets (cultured in chondrogenicor basal media) were used as an experimental control for thedsDNA characterization under the same conditions as the cell–hydrogel constructs. All samples were evaluated before (BC) andaer cryopreservation (AC). BC corresponds to the period up to14 days in culture, that is D1 corresponds to one day in cultureaer the experiment started while D14 refers to the days hASCs–kCR were in culture before cryopreservation. AC corresponds tothe time immediately aer cryopreservation (D0) followed by anadditional 7 days in culture (D7). All samples were subjected tolight microscopy observation, DMA assay, live and dead-assay,DNA quantication assay, and histology and RT-PCR analysis(Table 1).

Samples analysed in each characterization were based on 4independent experiments.

CharacterizationMorphology and mechanical properties

The full structure and morphological characteristics of thedeveloped discs with encapsulated cells (cultured in CM or BM)and without cells (cultured only in BM) both before (BC) andaer cryopreservation (AC) were observed under an invertedlight microscope (Zeiss, Axiovert 40 PG-HITEC) equipped with adigital camera (Axiocam MRc5, Zeiss).

The mechanical properties of 1.5% (w/v) ionic kCR hydrogeldiscs BC and AC were characterized by DMA (TRITEC Admin8000B, Triton Technology). The viscoelastic measurements werecarried out at 37 �C in 5% KCl and the samples were immersedin the culture medium until performing the assay. Aer equil-ibration, the DMA spectra were obtained during a frequencyscan in the range of 0.1–10 Hz. The experiments were performedunder constant strain amplitude (50 mm). A small preload wasapplied to each sample to ensure that the entire disc surface wasin contact with the compression plates before testing and thedistance between plates was equal for all samples being tested.At least ve samples were tested for each condition.

Viability and proliferation of the encapsulated human adiposederived stem cells

Live and dead assays using uorescence labeling were used toassess the viability of the cells. In short, samples were collected,placed in a 48-well plate, and washed with PBS. Subsequently,500 mL of 1 mg mL�1 calcein AM in PBS (1 : 500, C3099, Invi-trogen) was added to each sample for 10 min at 37 �C and laterwashed with sterile PBS. Aerwards, the samples were incu-bated with 300 mL of 1.5 mM propidium iodide in PBS (PI;


1 : 1000, P1304MP, Invitrogen) for 5 min at room temperature,prior to microscopic analysis (Reected/Transmitted lightMicroscope, Axioimager Z1M, Zeiss).

DNA quantication was performed using the uorimetricPicoGreen double-stranded DNA assay according to the manu-facturer’s instructions (Quant-iT� PicoGreen� dsDNA Kit,P7589, Invitrogen). Briey, cell–hydrogel constructs, collected ateach time period, were rinsed twice in PBS to completely removethe culture medium, placed in a microtube containing 1 mLultra-pure water, and stored at�80 �C to induce the lysis of cellsby osmotic and thermal shock, respectively. Then, samples werethawed and the supernatant collected for the dsDNA quanti-cation assay. The uorescence of the samples was measuredwith a microplate reader (Synergie HT, Izasa) at Ex 480 nm andEm 520 nm. The DNA concentration was calculated using astandard curve (DNA concentration ranging from 0–2 mg mL�1)relating to the amount of dsDNA to the uorescence intensity.

Histological evaluationTypical proteoglycans and H&E staining

Samples collected before and aer cryopreservation were xedwith 3.7% (v/v) neutral buffered formalin solution and stored at4 �C until analysis. Samples were further processed by a series ofdehydration steps, embedded in paraffin (Microm EC350-2,Thermo Scientic) and sectioned at 3 mm (Microm HM 355,Thermo scientic). Toluidine Blue (TB) staining was used toevaluate the deposition of glycosaminoglycans (GAGs), acomponent of cartilage ECM. TB (T3260, Sigma) coloration wasperformed by dipping the cell laden hydrogel sections for 2–3 sin the staining solution. Aerwards, the stain was poured offand the samples washed, dehydrated, cleared in xylene(1.08681.1000, VWR), and mounted for further analysis. Forhaematoxylin and eosin (H&E) staining, aer hydration, samplesections were colored with Papanicolaou Harris hematoxylin(05-12011/L, Bio-optica) for 3 minutes, washed in running tapwater, and aerwards a blue stain enhancement was performedby immersion in 0.5% ammonium hydroxide solution (05002,Sigma) for 5–10 seconds. The sections were washed in runningtap water and stained in Eosin-Y (05-M10003, Bio-optica) for 30seconds. Finally, slides were dehydrated through series ofalcohol immersions from 30 until 100% (v/v) alcohol. Thestained cells were visualized under a light microscope andimages were taken with a digital camera (Axion MRc5, Zeiss).Slides were obtained from multiple cell–hydrogel constructs. Ineach construct different sections were selected in order toanalyze the homogeneous distribution of the cells and thematrix within the construct according to Table 1.

Immunolocalization of type I and II collagens

Fixed samples with 3.7% (v/v) formalin solution were paraffinembedded and sectioned. Before removing the paraffin, theslides were warmed, and the antigen retrieval was performed for20 minutes at 95 �C using 10 mM citrate buffer. Aerwards thesections were washed in PBS, and endogenous peroxidaseactivity was quenched with 0.3% hydrogen peroxide (31642,

Soft Matter, 2013, 9, 875–885 | 883

Table 2 Primers used for RT-PCR analysis and expected sizes of the PCR productsa

Target gene Ascension no. Primer forward Primer reverse Amplicons Tm [�C]

Aggrecan NM_001135 50TGA GTC CTC AAG CCT CCT GT 50CAG TGG CCC TGG TAC TTG TT 171 bp 60.4Collagen II NM_033150 50GGG AGT AAT GCA AGG ACC AA 50ATC ATC ACC AGG CTT TCC AG 175 bp 57.4SOX9 NM_000346 50TAC GAC TAC ACC GAC CAC CA 50CTC CTC AAG GTC GAG TGA GC 217 bp 56.2Collagen X NM_000493 50CAG GCA TAA AAG GCC CAC TA 50AGG ACT TCC GTA GCC TGG TT 179 bp 57.4Collagen I NM_000089 50AGC CAG CAG ATC GAG AAC AT 50ACA CAG GTC TCA CCG GTT TC <200 bp 51.5GAPDH NM_002046 50TGC ACC ACC AAC TGC TTA GC 50GGC ATG GAC TGT GGT CAT GAG 87 bp 60.6

a bp – Base pairs and GAPDH – glyceraldehyde-3-phosphate dehydrogenase.

Soft Matter Paper

Sigma) for 5–15 min. Sections were washed with PBS andblocked with 2.5% horse serum from R.T.U. Vectastain�Universal Elite ABC Kit (PK-7200, Vector Laboratories) for 1 h toavoid nonspecic staining and further incubated with primaryantibodies (mouse anti-collagen II antibody – MAB1330, Milli-pore, and rabbit anti-collagen I antibody – ab292, Abcam)overnight at 4 �C, in a humidied atmosphere. Aerwards theslides were incubated with secondary antibody (R.T.U. Vectas-tain� Universal Elite ABC Kit) for 1 h at room temperature anddeveloped with the DAB substrate kit for peroxidase (SK-4100,Vector Laboratories). Slides were counterstained with haema-toxylin, mounted and visualized under the light microscope.Controls were performed using normal horse serum replacingthe primary antibodies.

RNA isolation and gene expression analysis (RT-PCR)

RT-PCR analysis was performed to detect the gene expression oftypical markers for chondrogenic differentiation, namelyaggrecan, collagen type II, SOX9, collagen type X and collagentype I. Total RNA was extracted using TRI Reagent� RNAIsolation Reagent (T9424, Sigma) following the manufacturer’sinstruction. First-strand complementary DNA was synthesizedfrom 2 mg of RNA of each sample reverse transcribed (qScript�cDNA Synthesis Kit, Quanta Biosciences) in a 20 mL reactionusing an Eppendorf Mastercycler� ep realplex gradient Smachine. mRNA expression of the genes of interest wasmeasured in the hASCs laden kCR hydrogels as well as in thepellet culture systems, both before and aer cryopreservation.The transcript expressions of target genes were analyzed to theexpression of endogenous housekeeping gene GAPDH (glycer-aldehyde-3-phosphate dehydrogenase). The primer sequencewas obtained from Primer3 soware (v 0.4.0), and synthesizedby MWG Biotech (see Table 2). Conventional PCR was per-formed using a High-Fidelity DNA Polymerase (Finnzymes) anddNTP Mix (Finnzymes) as well as a primer concentration of0.2 mM in a 35 cycle 3-step reaction. The PCR products weredetected in a 1.3% agarose gel (SeaKem�LE agarose, Lonza)using a 100 bp DNA ladder (GeneRuler� SM024, Fermentas).Images from the gels were taken using a UV Transilluminator(BioSpectrum AC Chemi HR 410, UVP).

Statistic analysis

All data values are presented as mean � standard deviation.Differences were determined to be statistically signicant with

884 | Soft Matter, 2013, 9, 875–885

p < 0.05. For statistic analysis one-way ANOVA analysis of vari-ance between groups with the Tukey post test was carried out.

Conclusions

This study evidences that complex tissue engineered productsaimed at clinical needs can be stored and preserved through thecryopreservation process. In the proposed system, it wasobserved that k-carrageenan hydrogels pledged structuralstability at a macro-scale and integrity with or without encap-sulated hASCs, registering also suitable mechanical propertiesaer the cryopreservation–thawing process. Also, encapsulatedhASCs maintained cell viability and chondrogenic featuresupon cryopreservation by restoring cellular metabolic func-tionalities 1 week of cell culturing aer thawing the constructs.The natural polymer k-carrageenan hydrogel seems to be resil-ient to the cryopreservation method used, and does notnegatively interfere with hASCs chondrogenic potentialdemonstrating its promising application as a cell carrier agentin “off-the-shelf” engineered tissue products. We envisionfuture studies applying these systems for transplantationfollowing an in vivo cartilage regeneration strategy.

Acknowledgements

We acknowledge nancial support to the Portuguese Founda-tion for Science and Technology (FCT) for the PhD grant SFRH/BD/64070/2009 (EP) and to the project MIT-Portugal Program(MIT/ECE/0047/2009).

References

1 L. Ji, J. J. de Pablo and S. P. Palecek, Biotechnol. Bioeng., 2004,88, 299–312.

2 W. Ma, T. O’Shaughnessy and E. Chang, Neurosci. Lett., 2006,403, 84–89.

3 B. C. Heng, L. L. Kuleshova, S. M. Bested, H. Liu and T. Cao,Biotechnol. Appl. Biochem., 2005, 41, 97–104.

4 M. C. Phelan, in Current Protocols in Cell Biology, John Wiley& Sons, Inc., 2001.

5 X. Zhang, P. N. Catalano, U. A. Gurkan, I. Khimji andU. Demirci, Nanomedicine, 2011, 6, 1115–1129.

6 F. Chen, W. Zhang, W. Wu, Y. Jin, L. Cen, J. D. Kretlow,W. Gao, Z. Dai, J. Wang, G. Zhou, W. Liu, L. Cui andY. Cao, Biomaterials, 2011, 32, 8426–8435.


Paper Soft Matter

7 G. Bhakta, K. H. Lee, R. Magalh~aes, F. Wen, S. S. Gouk,D. W. Hutmacher and L. L. Kuleshova, Biomaterials, 2009,30, 336–343.

8 L. H. Campbell and K. G. Brockbank, In Vitro Cell. Dev. Biol.:Anim., 2007, 43, 269–275.

9 S. Thirumala, J. M. Gimble and R. V. Devireddy, Stem CellsDev., 2010, 19, 513–522.

10 L. L. Kuleshova, S. S. Gouk and D. W. Hutmacher,Biomaterials, 2007, 28, 1585–1596.

11 A. Lawson, H. Ahmad and A. Sambanis, Cryobiology, 2011,62, 115–122.

12 Y. Miyamoto, S. Enosawa, T. Takeuchi and T. Takezawa, CellTransplant., 2009, 18, 619–626.

13 Y. Nie, V. Bergendahl, D. J. Hei, J. M. Jones and S. P. Palecek,Biotechnol. Prog., 2009, 25, 20–31.

14 R. Malpique, L. M. Osorio, D. S. Ferreira, F. Ehrhart, C. Brito,H. Zimmermann and P. M. Alves, Tissue Eng., Part C, 2010,16, 965–977.

15 H. Zimmermann, F. Ehrhart, D. Zimmermann, K. Muller,A. Katsen-Globa, M. Behringer, P. J. Feilen, P. Gessner,G. Zimmermann, S. G. Shirley, M. M. Weber, J. Metze andU. Zimmermann, Appl. Phys. A: Mater. Sci. Process., 2007,89, 909–922.

16 K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101, 1869–1879.

17 M. W. Tibbitt and K. S. Anseth, Biotechnol. Bioeng., 2009, 103,655–663.

18 A. D. Augst, H. J. Kong and D. J. Mooney, Macromol. Biosci.,2006, 6, 623–633.

19 E. Popa, R. Reis and M. Gomes, Biotechnol. Appl. Biochem.,2012, 59, 132–141.

20 J. M. Estevez, M. Ciancia and A. S. Cerezo, Carbohydr. Polym.,2008, 73, 594–605.

21 M. R. Mangione, D. Giacomazza, D. Bulone, V. Martoranaand P. L. San Biagio, Biophys. Chem., 2003, 104, 95–105.

22 E. G. Popa, M. E. Gomes and R. L. Reis, Biomacromolecules,2011, 12, 3952–3961.

23 R. C. Pereira, M. Scaranari, P. Castagnola, M. Grandizio,H. S. Azevedo, R. L. Reis, R. Cancedda and C. Gentili, J.Tissue Eng. Regener. Med., 2009, 3, 97–106.

24 V. E. Santo, A. M. Frias, M. Carida, R. Cancedda,M. E. Gomes, J. F. Mano and R. L. Reis, Biomacromolecules,2009, 10, 1392–1401.

25 P. M. Rocha, V. E. Santo, M. E. Gomes, R. L. Reis andJ. F. Mano, J. Bioact. Compat. Polym., 2011, 26, 493–507.


26 A. Grenha, M. E. Gomes, M. Rodrigues, V. E. Santo,J. F. Mano, N. M. Neves and R. L. Reis, J. Biomed. Mater.Res., Part A, 2009, 92, 1265–1272.

27 G. R. Diaferia, S. S. Dessi, P. Deblasio and I. Biunno,Cytotechnology, 2008, 58, 11–16.

28 P. F. Costa, A. F. Dias, R. L. Reis and M. E. Gomes, TissueEng., Part C, 2012, 18, 852–858.

29 K. G. M. Brockbank, J. R. Walsh, Y. C. Song and M. J. Taylor,in Topics in Tissue Engineering, ed. P. Ferretti and N.Ashammakhi, 2003.

30 D. J. McHugh, Production and Utilization of Products fromCommercial Seaweeds, Food and Agriculture Organizationof the United Nations, Rome, 1987.

31 H. A. Awad, M. Quinn Wickham, H. A. Leddy, J. M. Gimbleand F. Guilak, Biomaterials, 2004, 25, 3211–3222.

32 E. Afrimzon, N. Zurgil, Y. Shafran, F. Ehrhart, Y. Namer,S. Moshkov, M. Sobolev, A. Deutsch, S. Howitz,M. Greuner, M. Thaele, I. Meiser, H. Zimmermann andM. Deutsch, BMC Cell Biol., 2010, 11, 83.

33 A. De Rosa, F. De Francesco, V. Tirino, G. A. Ferraro,V. Desiderio, F. Paino, G. Pirozzi, F. D’Andrea andG. Papaccio, Tissue Eng., Part C, 2009, 15, 659–667.

34 A. M. Bhosale and J. B. Richardson, Br. Med. Bull., 2008, 87,77–95.

35 M. D. Kofron, N. C. Opsitnick, M. A. Attawia andC. T. Laurencin, J. Orthop. Res., 2003, 21, 1005–1010.

36 I. Sekiya, K. Tsuji, P. Koopman, H. Watanabe, Y. Yamada,K. Shinomiya, A. Nifuji and M. Noda, J. Biol. Chem., 2000,275, 10738–10744.

37 M. Locke, V. Feisst and P. R. Dunbar, Stem Cell, 2011, 29,404–411.

38 K. Pelttari, E. Steck and W. Richter, Injury, 2008, 39, 58–65.39 E. Mui~nos-Lopez, M. E. Rendal-Vazquez, T. Hermida-Gomez,

I. Fuentes-Boquete, S. Dıaz-Prado and F. J. Blanco, OpenOrthop. J., 2012, 6, 150–159.

40 M. K. Mamidi, K. G. Nathan, G. Singh, S. T. Thrichelvam,N. A. N. My, N. A. Fakharuzi, Z. Zakaria, R. Bhonde,A. K. Das and A. S. Majumdar, J. Cell. Biochem., 2012, 113,3153–3164.

41 P. C. Baer and H. Geiger, Stem Cells Int., 2012, 2012, 11.42 I. Reiter, M. Tzukerman and G. Maor, Bone, 2002, 31, 333–

339.43 G. Liu, H. Zhou, Y. Li, G. Li, L. Cui, W. Liu and Y. Cao,

Cryobiology, 2008, 57, 18–24.44 T. Rada, R. L. Reis andM. E. Gomes, Tissue Eng., Part B, 2009,

15, 113–125.

Soft Matter, 2013, 9, 875–885 | 885

Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies

Documents

Transcript of Cryopreservation of cell laden natural origin hydrogels for cartilage regeneration strategies