Thermally reversible polymer gel for chondrocyte culture

Angela Au,1 Jinny Ha,1 Anna Polotsky,1 Karol Krzyminski,2 Anna Gutowska,2 David S. Hungerford,1

Carmelita G. Frondoza1

1Department of Orthopaedic Surgery, Johns Hopkins University, 5601 Loch Raven Boulevard, Baltimore,Maryland 212392Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, Washington 99352

Received 28 October 2002; revised 18 April 2003; accepted 5 May 2003

Abstract: We have evaluated a biomaterial to serve as ascaffold for the propagation and amplification of chondro-cytes that promotes the original cellular phenotype of thesecells. The goal of the present study was to investigate the useof thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the prop-agation of chondrocytic cells. The polymer gels at tempera-tures above its lower critical solution temperature whereasliquefying at temperatures below its lower critical solutiontemperature of 34.5°C. Hence, the polymer, in its gelledform, has the ability to hold cells in situ, forming a matrixsimilar to the natural cellular environment or the extracel-lular matrix that comprises cartilage. We tested the hypoth-esis that the polymer gel promotes cell viability and func-tion. Human osteoblast-like cells, nasal chondrocytes, andarticular chondrocytes (1 � 105/150 �L) were resuspendedin enriched Dulbecco’s minimal essential media and wereplated onto control (without gel) and gel containing 24-wellplates. The plates were reincubated at 37°C, 5% CO2 for thetime point of interest. Additional media was added to the

plates and exchanged as needed. After cell culture, cellswere retrieved, enumerated, and cell viability was deter-mined. Other aliquots of the cells were stained for morpho-logical analysis whereas expression of chondrocyte markersincluding collagen type II and aggrecan were determinedusing reverse transcriptase–polymerase chain reaction. Thepolymer gel was not cytotoxic because the cell numberretrieved from three-dimensional culture gel was found tobe one to two times higher than that retrieved from mono-layer culture. Chondrocytes propagated in the thermo-re-versible polymers expressed enhanced or maintained ex-pression of collagen type II and aggrecan. Collagen type Iexpression was decreased or unaltered. The N-isopropylac-rylamide and acrylic acid copolymer gel has potential use asa cell culture substrate and as a cell delivery vehicle. © 2003Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1310-1319,2003

Key words: polymer; chondrocyte; thermo-reversible tissueengineering; tissue culture

INTRODUCTION

The search for biomaterials that can serve as three-dimensional scaffolds for cell culture and delivery is amajor focus in the field of tissue engineering. Thecurrent challenge is to find suitable biomaterials thatwill serve as a scaffolding framework for cell propa-gation, and amplification, while maintaining the orig-inal cellular phenotype.1 They need to facilitate theproduction of components that simulate the constitu-ents of original tissue. However, the availability of

such biocompatible materials is limited and the ques-tion of which biomaterial is suitable for cell propaga-tion remains an unsolved challenge.

Current tissue engineering approaches have usedseveral different materials for cell propagation. Thusfar, scaffolds constructed of natural or synthetic bio-materials have been identified for use as a frameworkon which cells can grow and attach. Natural porousmaterials such as collagen-based resorbable scaffoldsand chitosan matrices have previously been tested asthree-dimensional scaffolds. Collagen-like materialsexhibit high biocompatibility, pore density, and bio-degradation.2 Appropriate pore density allows forseeding and propagation of cells whereas biodegrada-tion rate is controlled by crosslinking and hydrophi-licity.3 Other natural materials such as chitosan, de-rived from chitin, have also been studied.4 Chitosan iscomposed of bioactive polysaccharides that seem to bebiocompatible and promotes chondrocyte prolifera-tion.5,6 These natural materials serve as support for the

Correspondence to: C. G. Frondoza; e-mail: cgfrondo@jhmi.edu

Contract grant sponsor: Department of Energy Office ofBiological and Environmental Research and the Good Sa-maritan Hospital

Contract grant sponsor: Johns Hopkins University and theDepartment of Orthopedic Surgery, School of Medicine

© 2003 Wiley Periodicals, Inc.

production of tissue-like materials. They degrade overtime whereas cells continue to form matrix compo-nents. However, natural polymers such as collagenand chitosan may not offer appropriate mechanicalproperties required for cell scaffolds.7

To circumvent the limitations of materials derivedfrom natural products, other investigators exploredthe use of synthetic polymers and their hybrids. Syn-thetic, resorbable materials that have previously beenevaluated for cell scaffolding include biodegradablepolymers.8 Examples of these are poly(glycolic acid)and poly(L-lactic acid).9 Such synthetic polymers wereclaimed to exhibit suitable physico-mechanical prop-erties although they have been claimed to exhibit in-ferior biocompatibility.10–12 In addition, synthetic andnatural materials can be combined, resulting in a hy-brid material that displays features of both materials.These biodegradable polymers resorb to accommo-date cell multiplication, allowing cells to organize anddifferentiate in the scaffolds. Examples of such hybridsystems include poly(vinyl alcohol)/chitosan blendsand chitosan/poly(acrylamide) interpenetrating poly-mer networks.13,14 However, cells do not always ad-here securely to these hybrid materials because theyare highly hydrophobic and repel water. Cells withinsuch hybrid materials are not evenly dispersedthroughout the scaffold. Some areas of the scaffold aremore densely seeded with cells than others. Althoughattempts have been made to optimize these materialsfor cell seeding, it is difficult to find a balance betweenthe biocompatibility of the material and the mechani-cal requirements needed.15–17 It is thus imperative tofind suitable materials that are biocompatible andhave appropriate mechanical and physical propertiesthat do not compromise their ability to support cellamplification and matrix production.

Fully thermo-reversible gelling polymers are amongthe resorbable polymers that have been considered foruse as scaffolding materials. These thermo-reversiblepolymers can revert from a solid to liquid state andvice versa an unspecified number of times withoutlosing their intrinsic properties. They are fully solublein water and other aqueous solutions at temperaturesbelow their lower critical solution temperature(LCST). However, at temperatures above its LCST, thepolymer solidifies to form a hydrated gel. Newly syn-thesized reversibly gelling polymers have been eval-uated according to their characteristics and potentialapplications.18 Applications involving thermo-revers-ible gels include tissue culture with human chondro-cytes, use as three-dimensional scaffolds, and as celldelivery agents.19,20 Because the polymer gels at tem-peratures at or above body temperature, the polymerwill solidify when placed in simulated body condi-tions. The cells can be seeded in the polymer at tem-peratures below the LCST, or below body tempera-ture. The polymer is in a liquid state and cells can be

thoroughly mixed with the polymer. The cell-seededpolymer can then be placed at temperatures above theLCST to facilitate gelling and solidification. Cells canbe grown in a three-dimensional culture mimickingthe natural environment of the cells. Propagation ofcells in such biocompatible matrices inhibits their ten-dency to shift to a fibroblastic phenotype in vitro.Utilizing the thermo-reversible gel allows cells to re-main immobilized in situ so that the matrix can serveas a vehicle for cell delivery into the tissue defect.

Little research has been done to analyze the abilityof such thermo-reversible polymers to serve as cellscaffolds. We have started to evaluate thermo-revers-ible gels composed of copolymers of N-isopropylac-rylamide (NiPAAm) as potential candidate biomateri-als for tissue engineering. In the proposed study, wehypothesize that the poly(NIPA-co-AAc) thermally re-versible polymer gel can serve as a biocompatiblesupporting scaffold for the propagation of viable andfunctional chondrocytes. Availability of such materi-als may be of use in tissue engineering to repair car-tilage. The selection of an appropriate biomaterial thatwill allow propagation and amplification of cells whilepromoting the original cellular phenotype would be acritical achievement in the laboratory.

MATERIALS AND METHODS

Materials

N-isopropylacrylamide (NIPA) (Acros Organics, Pitts-burgh, PA) was recrystallized from n-hexane and dried un-der vacuum. Acrylic acid (AAc) (99%; Aldrich, Milwaukee,WI) and 2-(N,N-dimethylamino)-ethyl acrylate (DMAEA)(Polysciences, Warrington, PA) were distilled under re-duced pressure. 2,2�-Azobisisobutyronitrile (AIBN) (98%;Aldrich) was recrystallized from methanol [high-perfor-mance liquid chromatography (HPLC) grade; Aldrich]. Di-oxane (HPLC grade; Aldrich) was distilled under nitrogenand degassed before use. 2-Carboxyethyl acrylate (DAAc)(60%; Polysciences), benzene (anhydrous, 99.8%), diethylether, and hexane (reagent grade; Aldrich) were used asreceived.

Polymer synthesis

Ionic NiPAAm with AAc, DAAc, and DMAEA were syn-thesized by free-radical solution copolymerization in diox-ane/benzene mixtures. The reaction mixtures were purgedwith deoxygenated nitrogen for 0.5 h before the initiator,AIBN, was added. Polymerization was conducted undernitrogen at 70°C for 18 h. The resulting reaction mixture wascooled to room temperature, diluted with acetone, precipi-tated into diethyl ether, and the crude polymer precipitatewas filtered, washed with diethyl ether, and dried under

THERMO-REVERSIBLE GELLING POLYMERS 1311

vacuum. Dried polymers were dissolved in ultra-pure wa-ter, filtered through a nylon membrane (pore size 0.45 �m)and further purified using ultrafiltration cell (Amicon Inc.).Membranes with nominal molecular weight cut-off of 30,000and 300,000 were used for purification. In the final step,purified polymer solutions were freeze-dried.

Molecular weight determination

The weight-average molecular weight (Mw) and polydis-persity indexes (Mw/Mn) were determined using gel perme-ation chromatography. The gel permeation chromatographyset consisted of two Styragel� columns (HMW 6E and HR4E; Waters, Inc.), 410 Differential Refractometer (Waters,Inc.), 515 HPLC pump (Waters Inc.), and a miniDAWN lightscattering detector (Wyatt, Inc.). Tetrahydrofuran (HPLCgrade; Aldrich) was used as a mobile phase.

Dynamic rheometry

Rheological properties of solutions and gels were studiedusing an SR 2000 dynamic rheometer (Rheometric Scien-tific). Parallel plates with 25-mm diameter and 0.5-mm gapwere used in all experiments. Temperature-dependentchanges in elastic (storage) modulus, G�, and viscous (loss)modulus, G� were recorded in a dynamic temperature ramptest (DTRT). The DTRT was conducted under controlledstress of 2.0 Pa, frequency of 1.0 rad/s, and temperatureramp rate of 0.3°C/min with heating–cooling cycle set be-tween 20–50°C. The rheometric experiments were con-ducted using 10 wt % polymer solutions in phosphate-buffered saline or in water.

Screening of the polymers

Five different formulations of the NIPA copolymers wereinitially screened to determine the optimal thermal-revers-ible polymer for use as a scaffold (Table I). Three negativelycharged copolymers of the NIPA with 2 mol % of AAc,poly(NIPA-co-AAc), (K-63I, K-66, nd K-71) were tested. Twopositively charged copolymers of NIPA with 3 mol % ofDMAEA, poly(NIPA-co-DMAEA) (K-67 and K-68) were alsotested. Polymer composition, molecular weight, charge, andpolymer concentration in water [wt %] for each of the five

formulations are listed in Table I. The K-63I formulation wasinitially prepared in a 10 wt % solution in deionized water inan external ice bath using a magnetic stirrer. After polymerdissolution, the polymer solution was sterilized by autoclav-ing for 30 min and its pH adjusted to 7. The polymersolution was then cooled at or below the LCST of 34.5°C toensure complete liquefication of the gel before use.

Evaluation of cell viability and number

Human osteoblast tumor cells (MG-63) were used forcytotoxicity prescreen. Articular chondrocytes were isolatedfrom articular cartilage during primary knee replacements.21

Nasal cartilage was acquired from patients undergoing na-sal reconstructive surgery.22 The cartilage obtained wasdiced into 1-mm3 pieces. Digestion of the cartilage with 0.1%(w/v) collagenase (Boehringer Mannheim, Germany) for12 h at 37°C and 5% CO2, with constant stirring, isolated thechondrocytes. Washing the digested cartilage twice withHank’s balanced saline solution terminated enzymatic di-gestion. A Cellector™ tissue sieve separated individualchondrocytes from undigested cartilage and cartilage debris.

The trypan blue uptake method was used to enumeratecells using a hemacytometer. Stained cells that took up theblue dye were considered dead whereas the unstained cellswere considered viable. Phase-contrast microscopy wasused to study cells morphology.

Propagation of MG-63 human osteoblast-like cellsand chondrocytes in monolayer and three-dimensional culture

MG-63 osteoblast-like cells and chondrocytes were prop-agated and passaged for up to four passages (2 months) inmonolayer culture at 37°C and 5% CO2 until ready for use.Cells were harvested from cell culture with 0.25%trypsinization when confluent and were counted and as-sayed for viability using the trypan blue uptake method.Once the number of cells was assessed, the needed amountof chondrocytes at a density of 1 � 105 cells/150 �L werethoroughly resuspended in 50% HY medium made with 2XDulbecco’s minimal essential medium supplemented withfetal calf serum. The cell/media mixture (150 �L) was platedin each well on a 24-well plate. An additional 150 �L of HYmedium was added to wells serving as control (withoutgel-monolayer culture). An additional 150 �L of the K-63I

TABLE ICharacteristics of the Thermo-Reversible Polymer Gels Screened

Formulationwt % Solution in

Water mol % ChargeMolecular Weight

(kD)

K-63 poly(NIPA-co-AAc) 10 98/2.0 Negative 851.0 (�73.8)K-66 poly(NIPA-co-AAc) 20 98/2.0 Negative 235.3 (�14.1)K-67 poly(NIPA-co-DMAEA) 10 97.1/2.9 Positive 519.0 (�54.7)K-68 poly(NIPA-co-DMAEA) 10 97.1/2.9 Positive 354.0 (�12.8)K-71 poly(NIPA-co-AAc) 10 98/2.0 Negative 602.0 (�33.8)

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polymer gel was also added to the remaining wells so thateach cell/polymer/media suspension consisted of 300 �L ofprewarmed 1X media composed of no more than 4–5% (ofpolymer) by weight. The contents of each well were thor-oughly mixed with a pipette to ensure that the cells wereevenly suspended in the polymer solution and in the controlwells. The gels were immediately incubated for approxi-mately 1 h at 37°C and 5% CO2 to allow the polymer to forma gel. To ensure that the plates remained at �37°C at alltimes, an additional 1.0 mL of prewarmed media was gentlypipetted over the control wells and the solidified gels mak-ing sure the media did not dissolve the gelled matrix. Theplates were kept above 37°C to prevent liquefication of thepolymer gel and keeping the cell immobilized in situ. Addi-tional media was exchanged as needed throughout the timepoint of interest (3–14 days).

The cells from the monolayer culture were retrieved atapproximately 90% confluence by trypsinization. Cells fromthe three-dimensional gel culture were harvested by placingthe plates at room temperature for approximately 15 min tofacilitate the liquefaction of the polymer gel. One hundredmicroliters of cold Hank’s balanced saline solution wasadded to each well to further liquefy and dissolve the poly-mer gel to facilitate cell retrieval. Both gel-containing andmonolayer plates were trypsinized to detach any remainingcells. Additional Hank’s balanced saline solution was usedto wash each well. The cells were enumerated and assayedfor cell viability using the trypan blue uptake method. Ali-quots of the cells were made to assess cell viability andphenotype expression at the protein and at the mRNA level.Reverse transcriptase–polymerase chain reaction (RT-PCR)analysis was used to verify the presence of collagen type IIand gene-specific chondrocytic markers.

Determination of cell morphology

Aliquots of the chondrocytes were cytocentrifuged for 6min onto lysine-coated slides. The HEMA 3� Stain Set (Bio-chemical Sciences Inc.) was used to stain lysine-coatedslides. The three dip staining process produced slides exhib-iting high contrast and granular resolution.

RNA extraction and analysis by RT-PCR

Total RNA was isolated using the TRIzol� (Life Technol-ogies™, Rockville, MD) reagent method. Total cDNA librar-ies were synthesized, using 1 �g of total RNA, the Oligo(dT18) primer, and the advantage RT-PCR kit (ClontechLaboratories, Palo Alto, CA). The SuperTaq Plus (Ambion,Austin, TX) PCR kit and specific primers for collagen type IIand I, proteoglycans-aggrecan, and the housekeeping geneGAPDH or S14 ribosomal subunit expanded the resultingRT product.23

Each PCR reaction required the use of 2 �L of cDNA.Primers for collagen type II (sense: 5� CAC CTT GGA CGCCAT GAA GGT 3�; antisense: 5� GTG AAC CTG CTA TTGCCC TCT 3�), collagen type I (sense: 5� GAC GGG AGT TTCTCC TCG GGG TC 3�; antisense: 5� GAG TCT CCG GAT

CAT CCA CGT C 3�), and aggrecan (sense: 5� GGG TCAACA GTG CCT ATC AG 3�; antisense: 5� GGG TGT AGCGTG TAG AGA TG 3�) were used for amplification. PCRreactions for collagen type II, collagen type I, and aggrecanwere conducted in a Perkin-Elmer Thermal Cycler. Afterinitial treatment (75°C, 5� and 94°C, 1�), 30 of the followingcycles were performed: denaturation (94°C, 15�), annealing(65°C, 15�), and extension (68°C, 3�). Agarose gel electro-phoresis was used to analyze the PCR products, whereas theUN-SCAN-IT Gel Automated Digitizing System (Silk Scien-tific Corp.) was used for densitometry.

RESULTS

Polymer properties

The temperature-dependent properties of solutionsand gels were investigated using dynamic rheometrymethods. It was observed that aqueous solutions of allsynthesized copolymers flowed freely at room tem-perature and formed soft gels at physiological temper-ature range as a result of a sol–gel transition. The gelswere fully reversible, that is, exhibited also a gel–soltransition, and were classified as physical gels.

Temperature-dependent changes in G� and G� weremeasured using a DTRT. Results of a representativeDTRT are shown in Figure 1 for K63. During theheating cycle, H, a change in solution temperaturefrom 20° to 37°C resulted in dramatic increase of bothmoduli because of the temperature-driven sol–geltransition. The subsequent cooling cycle, C, resulted ingel-to-sol transition and fully reversible gel melting.The crossover of G� and G� heating curves correspondsto the gel formation temperature (GFT), and the cross-over of the cooling curves denotes the gel melting

Figure 1. Results of temperature-dependent changes in G�and G� measured by a DTRT. H is indicative of the heatingcycle from 20–37°C whereas C indicates the cooling cyclewithin the same ranges of temperature. Temperature-depen-dent changes in G� and G� moduli for K-63 copolymer,measured by a DTRT. The H curves denote the heating cycle(sol–gel transition), and C curves denote the cooling cycle(gel–sol transition). A 10 wt % solution of K-63 copolymer inphosphate-buffered saline, pH 7.4, was tested.

THERMO-REVERSIBLE GELLING POLYMERS 1313

temperature (GMT). The observed hysteresis loop,typical for polymeric networks, is a kinetically con-trolled phenomenon reflecting the resistance to disin-tegration of entangled polymer chains forming thehydrogel network.

Table I summarizes polymers used in the study. Thegelling time and degree of stiffness of the gels werefirst evaluated to determine the best formulation touse for further analysis. The K-63I poly(NIPA-co-AAc)copolymer with the highest molecular weight wasfound to be the best polymer formulation to use forfurther evaluation. The K-63 gels formed fast, had anappropriate stiffness, and also liquefied rapidly forefficient cell recovery. The optimal properties of theK-63 resulted from the high molecular weight of thispolymer.

Articular and nasal chondrocyte growthcharacteristics in monolayer and three-dimensionalculture

Initial prescreening of the K-63I polymer gel usingosteoblast-like cells indicated that this polymer is notcytotoxic. The percent cell viability of MG-63 con-firmed that the copolymer was nontoxic and could befurther evaluated using chondrocytes (Fig. 2). Afterprescreen, chondrocytes were propagated in gel andmonolayer culture from 3 to 14 days. Throughout theincubation period, cells in monolayer culture ap-peared to adhere readily to the bottom of the plates.The growth of cells propagated in monolayer culture,which appear to parallel increase in cell number, couldbe monitored using a light microscope. At 90% con-fluence, cells were stretched out and became moredensely packed. However, growth of cells cultured inthe polymer gel could not be determined using a lightmicroscope, because the polymer solution turnedopaque upon gelling and the cells could not be seen.

Hence, it was difficult to determine whether or not thecells propagated in the gel once the polymer solidified,until the time of final cell count.

When propagated in the K-63I gels, both articularand nasal chondrocytes remained viable. Chondrocytecell lines isolated from articular and nasal cartilagewere approximately 80% viable for up to 2 weeks inculture in the K-63I polymer gel. The number of viablecells propagated in the three-dimensional gel was ap-proximately the same as those propagated in mono-layer culture. The percent viability of articular chon-drocytes propagated in the gel and in the monolayerculture was 70–93% [Fig. 3(a)] whereas the viabilityfor nasal chondrocytes propagated in the two types ofculture was between 60–93% [Fig. 3(b)]. The figuresshowing graphs of cell viability plotted for differentcell lines of articular and nasal chondrocytes confirmthat cells remain viable in both monolayer and gelcultures. As shown in Figure 3, cell viability for all celltypes is not impaired by the presence of the thermo-reversible polymer gel.

The number of cells retrieved from the polymer gelranged from one to two times the number of viablecells retrieved from monolayer culture, as indicated byFigure 4(a,b). The total number of articular and nasalchondrocytes enumerated from monolayer cultureranged from 0.38 � 105 to 1.86 � 105 cells. In compar-ison, the yield of cells retrieved from the three-dimen-

Figure 3. Percent cell viability of different cell lines of (a)articular chondrocytes and (b) nasal chondrocytes retrievedfrom monolayer and three-dimensional (gel) culture. P3 �[Passage 3]; A1 � [Articular Chondrocyte Line 1]; N1 �[Nasal Chondrocyte Line 1].

Figure 2. Percent cell viability of different types of MG-63osteoblast-like cells, articular chondrocytes, and nasal chon-drocytes for initial prescreen of the K-63 formulation of thethermo-reversible polymer gel which showed that the gelwas not cytotoxic and could be used for further analysis.

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sional polymer gel ranged from 0.75 � 105 to 1.85 �105 cells, for both articular and nasal chondrocyte celllines. The number of articular chondrocytes propa-gated in the three-dimensional gel was significantlyhigher than those retrieved from monolayer culture(p � 0.05) [Fig. 4(a)]. In contrast, there was no statis-tical difference in the number of cells recovered fromthe gel and monolayer cultures [Fig. 4(b)]. Total aver-age cell numbers were sometimes less than theamount plated because cells may have been lost dur-ing retrieval.

Chondrocytes appeared to divide at least once inboth monolayer and gel-containing cultures; however,the longest time point that was achieved with thenumber of cells plated per well was 14 days (2 weeks).If the time points of interest were longer than 14 days,the cells in monolayer culture became overgrown andthe cells began to die. Evaluation of the cells using thelight microscope showed cells cultured in the mono-layer to be refractile and round. When the cells be-came more confluent, the cells that adhered to thebottom of the well became more elongated. Cells inthe gel could not be seen with the light microscopebecause the gel turned opaque upon solidification.Cell proliferation could only be determined by looking

in the monolayer culture and was used to determineconfluency of the cells in both cultures.

After retrieval, cells were cytospun and fixed usingthe HEMA 3� Stain Set to visualize the cell morphol-ogy using phase-contrast microscopy. Photomicro-graphs taken of articular chondrocytes after 3 days ofculture were shown with cells grown in monolayerand thermo-reversible gel culture. Cells propagated inmonolayer and in three-dimensional culture bothshowed spherical cells with smooth outlines with sim-ilar morphology [Fig. 5(a,b)]. The nuclei of each cellcould be distinguished in both types of culture. Noobvious difference could be seen between cells prop-agated in monolayer and cells propagated in the poly-mer gel. The bottom row of Figure 3 shows cellscultured for 7 days that showed different morphologybetween cells grown in monolayer and gel-containingcultures. Cells grown in monolayer culture were notas spherical shaped compared with cells grown in thethree-dimensional gel [Fig. 5(c,d)]. A fuzzy halo orring-like appearance also appeared to envelop thecells retrieved from the gel. The ring may be a result ofresidual polymer gel that did not completely wash offduring cell retrieval or to the synthesis of an extracel-lular matrix by the cells.

Evaluation of chondrocyte phenotype

RT-PCR analysis of mRNA levels for collagen type I,II, and aggrecan

The RT-PCR profiles display the phenotype expres-sion of three representative articular chondrocyte celllines propagated in control monolayer or gel cultures(Fig. 6). The top row confirms equal loading becausethe GAPDH housekeeping gene was of equal intensityfor both cultures [Fig. 6(a)]. Collagen type I and IImRNA levels from cells grown in the gel were com-parable to that of cells from monolayer culture [row2,3; Fig. 6(a)]. Aggrecan mRNA levels were upregu-lated in chondrocytes propagated in the gel [row 4;Fig. 6(a)]. The other two RT-PCR profiles also indi-cated that there was equal loading of the gel andnon-gel containing wells based on S14 housekeepinggene expression [row 1; Fig. 6(b,c)]. The phenotypeprofiles for Figure 6(b,c) indicated that the cell linesshowed similar expression of extracellular matrixcomponents produced by chondrocytes. The secondrow of Figure 6(b) showed similar mRNA levels ofcollagen type I. In contrast, collagen type II expressionwas enhanced in cells retrieved from gels than frommonolayer culture [row 3, Fig. 6(b,c)]. Similar levels ofaggrecan transcripts were observed in cells propa-gated in the presence and absence of the polymer [row4, Figure 6(b,c)].

Figure 4. Average cell numbers of different cell lines of (a)articular chondrocytes and (b) nasal chondrocytes retrievedfrom monolayer and three-dimensional (gel) culture. P3 �[Passage 3]; A1 � [Articular Chondrocyte Line 1]; N1 �[Nasal Chondrocyte Line 1].

THERMO-REVERSIBLE GELLING POLYMERS 1315

DISCUSSION

Of the five formulations of the thermo-reversiblegelling polymers screened, the K-63I formulation ofNIPA and AAc proved to be the most suitable to serveas a possible scaffolding material to support cell prop-agation. These gels were thermally reversible becausethey were able to solidify and liquefy an unspecifiednumber of times without losing their intrinsic proper-ties. Exhibiting LCST behavior at 34.5°C, the copoly-mer is fully soluble in water at temperatures below theLCST. However, when placed at temperatures abovethe LCST, the aqueous solution solidifies and turnsinto a soft, hydrated gel. Its ease of use, short gellingand melting time, ability to maintain its shape, and gelstiffness at temperatures above its LCST of 34.5°C,made it a candidate biomaterial to evaluate for use asa reversibly gelling matrix to propagate the cells. Itsgelling time was �5 min and the gel could be platedeasily. Gels with a lower modulus are able to liquefymore quickly, facilitating cell retrieval. In contrast,gels with a higher modulus are better able to maintaintheir shape when additional medium is added so thatcells are kept in place within the material. The K-63Ipolymer displays an appropriate modulus which al-lowed the gel to dissolve easily for cell retrieval butwas also adequate in holding the cells within thematrix.24–28 It dissolved at a slower rate than the otherformulations when additional media was pipetted ontop, without disrupting the cells in situ. In its gelledform, the K-63I polymer gel was sufficiently stiff towork with because it did not dissolve easily when

additional media was added at temperatures above itsLCST.

The thermo-reversible polymer gel as a scaffoldingmaterial exhibits properties similar to natural, porousmaterials such as alginate, chitosan, and dextran,which are examples of regenerated natural polysac-charides and their derivatives. Alginate systems arehighly branched and porous, allowing cells to growand propagate easily throughout the system.29 Whenthe thermo-reversible polymer used in the presentstudy is in its gelled, solidified form, a system similarto the alginate system is created as the cells are able tomultiply more readily between the porous areas. Incontrast, cells propagated in a more rigid gel wouldhave a more limited ability to multiply through the gelmatrix. More rigid gels such as systems made of col-lagen are considerably stiffer and have highly struc-tured three-dimensional matrices. These cells are notable to move around freely in such constrained envi-ronments.

Cells propagated in the poly(NIPA-co-AAc) gel-con-taining culture were found to have an enhancedgrowth rate compared with cells grown in a mono-layer culture. Although it was presumed that the gelmatrix would hinder the cells from propagatingthrough the scaffold, cells did proliferate at a greaterrate in the three-dimensional culture. The gel allowedfor more surface area where the cells could multiply inmore than just one layer. The cells appear to haveproliferated significantly throughout the time point ofinterest, which ranged from 3 to 14 days, as shown bythe doubling in the number of cells propagated in the

Figure 5. Phase contrast photomicrographs showing cell morphology of human articular chondrocytes retrieved after (a) 3days in monolayer culture, (b) 3 days in gel-containing culture, (c) 7 days in monolayer culture, and (d) 7 days ingel-containing culture on lysine-coated slides by cytocentrifugation. The cells have been stained using the HEMA 3� Stain Setand appear spherical with distinct nuclei. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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gel compared with monolayer cultures. The amount ofcells plated was very close to the number of cellsretrieved after culture and were attributed to variablesincluding the exchange of media, the length of timethe cells were cultured, and the possibility of the gelmelting during replacement of the media. Hence, thegel boosts and improves the rate of cell proliferation.

Our observations show that not all cells behaved thesame while in the gel. Differences in cell behaviordepended on the type of cells used, passage number,and duration the cells were in culture before seedingin the gel and monolayer cultures. Older cells, or cells

which had been passaged several times, may havealready been fibroblastoid, causing a shift in pheno-type before use in the study. Several experimentsneeded to be terminated earlier than the set time pointof interest because some of the cell lines grew fasterthan others. If these cells were not retrieved fromculture before overcrowding occurred, premature celldeath may have resulted, leading to decreased cellviability although the number of cells retrieved mayhave remained high. In addition, there was a markedchange in cell morphology after 7 days in culturewhich may have been attributed to possible over-

Figure 6. (Three days) RT-PCR phenotype gel for human articular chondrocytes for demonstration of collagen types I andII and aggrecan. Chondrocytes harvested from the monolayer and gel culture at 90% confluence were frozen at 70°C. RNAextraction was performed for RT-PCR analysis. Three different articular chondrocyte cell lines were used and are shown in(a), (b), and (c) lines 1, 2, and 3, respectively.

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crowding of cells in the monolayer cultures. Cells inthe gel, however, could grow in more than one plane.Therefore, overcrowding did not occur as easily andthose cells could grow more freely.

The RT-PCR profile in Figure 6(a) indicated that thecells had shifted to a fibroblastoid phenotype as col-lagen types II and I were coexpressed whereas aggre-can expression was enhanced in the K-63I polymergel. The increase in aggrecan expression indicated en-hanced expression of the biomarkers for the chondro-cytic phenotype.30 The shift to a fibroblastoid pheno-type indicates the cells may have been fibroblastoidbefore cell culture or that the polymer gel may haveshifted the phenotype. The presence of collagen type IIalso shows that the cells propagated in the polymergel maintained their phenotype. This may be attrib-uted to the constrained environment which the poly-mer gel evokes that closely mimics the natural state ofchondrocytes in cartilage. Thus, this “natural” envi-ronment may have stimulated the synthesis the orig-inal products of collagen type II and aggrecan foundin chondrocytes. The other two RT-PCR profiles, oftwo other articular chondrocyte cell lines, indicate thatthe thermo-reversible gel promotes the chondrocyticphenotype.

The cell culture method used in this study facili-tated the evaluation of candidate biomaterials for cellculture. The K-63I polymer gel supports cell viability,growth, and metabolic activity, allowing the copoly-mer to potentially be used as a reversibly gelling cellculture substrate. Our results provide evidence to sup-port our hypothesis that the thermally reversible poly-(NIPA-co-AAc) copolymer may be suitable as a cellculture substrate and may have potential use for tissuerepair. Researchers have implemented the use of sim-ilar thermally reversible hydrogels as injectable scaf-folds for tissue engineering applications.31,32 Furtherstudies are being performed to fully optimize the for-mulation of thermally reversible gels for use in chon-drocyte culture and as a cell delivery vehicle.

The authors thank Dr. Alan Shikani for supplying dis-carded nasal cartilage tissue and Shirley Anderson for herhelp in the preparation of the manuscript.

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