Technical Note

Evaluation of thermoreversible polymers containing fibroblast growthfactor 9 (FGF-9) for chondrocyte culture

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

Carmelita G. Frondoza1

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

Received 31 July 2003; revised 10 November 2003; accepted 18 November 2003Published online 25 February 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.20132

Abstract: We previously evaluated a thermoreversiblepolymer gel composed of N-isopropylacrylamide andacrylic acid as a cell culture substrate and cell-delivery ve-hicle. The copolymer promoted phenotype expression andamplification of chondrocytes. In this study, we determinedwhether addition of fibroblast growth factor 9 (FGF-9),which is mitogenic for chondrocytes, would further enhancecell proliferation and phenotype expression in the polymer.We tested the hypothesis that the thermoreversible polymercontaining FGF-9 would promote increased chondrocyteproliferation and phenotype expression. Articular chondro-cytes (1 � 105/150 �L) were plated onto control (withoutgel) and gel containing 24-well plates. The gels were pre-pared in media alone or in media containing heparin (100�g/mL) and FGF-9 (5 �g/mL). The cultures were incubated

at 37°C in 5% CO2 for 3 days. Cells remained viable in thethermoreversible polymer in the presence or absence ofFGF-9. Addition of FGF-9 to the copolymer did not induceproliferation and the cell numbers did not increase. Reversetranscription polymerase chain reaction (RT-PCR)-deter-mined expression of chondrocyte markers collagen type IIand aggrecan. FGF-9 did not enhance chondrocyte prolifer-ation nor alter the phenotype after 3 days in culture. Thesefindings suggest the poly(NiPA-co-AAc) gel alone may pro-vide the optimal 3D environment for propagation of chon-drocytes. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res69A: 367–372, 2004

Key words: chondrocytes; thermoreversible polymer; tissueengineering; growth factors; scaffold

INTRODUCTION

There is an ongoing search for suitable biomaterialsthat can serve as 3D scaffolds for cell culture anddelivery. These biomaterials need to promote cell vi-ability and synthesis of cellular products. Current ap-plications involving cell-based therapy for cartilagerepair requires scaffolding biomaterials that will facil-itate the amplification of cells in vitro while supportingthe production of extracellular matrix (ECM) compo-nents.1 The goal for these cell scaffolding materials isto facilitate the formation of cartilage-like tissue rather

than fibrocartilage. The question of which biomaterialto use for cartilage repair, however, remains a chal-lenge. The use of resorbable, synthetic materials com-posed of biodegradable polymers as cell scaffold ma-terials are currently being explored. These polymersenable cells to proliferate without altering their phe-notype.2 In particular, fully thermoreversible gellingpolymers have been studied for use as a scaffold tohold cells in situ. These thermoreversible polymers areable to revert from a solid to liquid state and vice versawithout losing their intrinsic properties.3 At tempera-tures below their lower critical solution temperature(LCST) these polymers are fully soluble in aqueoussolutions and at temperatures above their LCST solid-ify to form a hydrated gel.

The LCST of many thermoreversible polymers is 37°C,which is the normal human body temperature, and thepolymer solidifies above this temperature. Thus, cellscan be suspended in the polymer at temperatures belowits LCST and then placed at temperatures above theLCST for the cell–polymer suspension to solidify. Thepolymeric scaffold is able to hold cells in situ and creates

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

Contract grant sponsor: Department of Energy Office ofBiological and Environmental Research

Contract grant sponsor: Johns Hopkins University Schoolof Medicine

Contract grant sponsor: Johns Hopkins University Depart-ment of Orthopaedic Surgery

© 2004 Wiley Periodicals, Inc.

a 3D matrix mimicking the natural cellular environmentin vitro. Chondrocytes propagated in these matrices donot shift to fibroblastoid phenotypes. Thus, the cell-seeded 3D matrix can serve as a vehicle for cell deliveryinto the tissue defect.

We previously evaluated a thermoreversible copo-lymer comprised of N-isopropylacrylamide (NiPAAm)and acrylic acid (AAc) as a candidate scaffolding mate-rial.4,5 The K-63I formulation of the poly(NiPA-co-AAc) best supported chondrocyte viability, growth,and metabolic activity. In this study, we further eval-uated whether the K-63I thermoreversible polymer,with the addition of fibroblast growth factor 9 (FGF-9),would improve cell division and the production ofECM components. Previous studies have shown thatmembers of the fibroblast growth factor (FGF) familypromote chondrocyte proliferation as well as synthe-sis of the ECM.6,7 Moreover, growth factors have beennoted to modulate the development, proliferation, andmaintenance of skeletal tissues by the transforminggrowth factor-� superfamily (TGF-�), insulin-likegrowth factors (IGFs), hepatocyte growth factor(HGF), bone morphogenic proteins (BMPs), platelet-derived growth factors (PDGFs), and FGFs. The 22identified members of FGF, while having similar pro-tein structures, differ in their target specificity andexpression patterns. FGFs regulate mitogenesis, differ-entiation, and receptor expression in several types ofcells, including chondrocytes. Although FGF-1 (acidicFGF) and FGF-2 (basic FGF) have been well character-ized as chondrocyte mitogens, the effect of other mem-bers of the FGF family on chondrocytes is not as wellknown.8,9

The recently identified FGF-9 has been detected inpatients affected with hereditary multiple exostosisand primary synovial chondromatosis—both ofwhich are characterized by extra-articular cartilageformation also known as cartilaginous metaplasia. Sy-noviocytes taken from such patients have been docu-mented to produce FGF-9 while in culture. FGF-9shares 30% sequence identity with FGF-1 and FGF-2and was originally purified as a heparin-binding, glia-activating factor with a unique receptor specificity.Heparin has been shown to promote high-affinitybinding to its respective receptors. FGF-9 has beencharacterized as a specific, high-affinity ligand, depen-dent on heparin for binding to FGFR3.10–12 WhileFGF-9 binds with low affinity for FGFR2, it fails tobind with FGFR1 or FGFR4. Although FGF-9 has beenshown to stimulate chondrocyte proliferation, its ef-fect on the ability of these cells to form extracellularcartilage has not been evaluated.13–15 In this study, weevaluated whether the addition of FGF-9 furtherenhances chondrocyte proliferation in the thermo-reversible polymer while promoting expression of car-tilage-specific components. We hypothesize that ther-moreversible copolymers composed of NiPAAm and

AAc containing FGF-9 will serve as biocompatiblescaffolds that support viable and functional chondro-cytes.

MATERIALS AND METHODS

2.2�-Azobisisobutyronitrile (AIBN) (98%; Aldrich) was re-crystallized from methanol [high-performance liquid chro-matography (HPLC) grade, Aldrich]. Dioxane (HPLC grade;Aldrich) was distilled under nitrogen and degassed prior touse. N-isopropylacrylamide (NIPA) (Acros Organics) wasrecrystallized from n-hexane and dried under vacuum whileAAc (99%; Aldrich) and 2-(N,N-dimethylamino)-ethyl acry-late (DMAEA) (Polysciences) were distilled under reducedpressure. 2-Carboxyethyl acrylate (DAAc) (60%; Poly-sciences), benzene (anhydrous; 99.8%), diethyl ether, andhexane (reagent grade, Aldrich) were used as received.FGF-9 (100 ng/mL; R&D Systems) and heparin (2 �/mL;Sigma) were each diluted with Hanks Balanced Saline Solu-tion (HBSS) and sterile filtered prior to use.

The NiPAAm and AAc thermoreversible polymer wassynthesized through free radical copolymerization and theK-63I formulation was chosen as the optimal thermorevers-ible polymer to use as a scaffold. It is a negatively chargedcopolymer composed of NiPAAm and 2 mol % of AAc. TheK-63I formulation was synthesized in a 10% w/v solution indeionized water in an external ice bath using a magneticspinner. The polymer solution was sterilized by autoclavingfor 30 min and pH was adjusted to 7 following polymerdissolution. To ensure complete liquefaction of the polymerprior to use, the polymer solution was consequently cooledto or below its LCST of 34.5°C.16

Articular chondrocytes were isolated from articular carti-lage obtained from patients undergoing primary knee ar-throplasties. Sterile cartilage tissue was diced into 1-mm3

pieces and digested with 0.1% (w/v) collagenase A (Boehr-inger Mannheim, Germany) for 12 h at 37°C in 5% CO2 withconstant stirring. Enzymatic digestion was terminated bywashing the digested cartilage twice with HBSS. Undigestedcartilage and cartilage debris were separated from chondro-cytes using a Cellector tissue sieve.17 Chondrocytes werepropagated and passaged in T-75 flasks at 37°C in 5% CO2

for up to four passages (2 months). When confluent, cellswere harvested with 0.25% trypsin and enumerated andassayed for viability using the trypan blue uptake method.Cells were resuspended in 10% 2X HY medium made with2X Dulbecco’s minimal essential medium (DMEM) supple-mented with 10% fetal calf serum (FCS), 2% L-glutamine,and 0.05% gentamycin at a density of 1 � 105 cells/150 �Lper well (24-well plate) (150 �L). The cell/media suspensionwas plated in each well in two 24-well plates. Heparin (100�g/mL—final concentration in media) was added to allwells while FGF-9 (5 �g/mL—final concentration in media)was only added to half the wells in each plate. The concen-tration of the FGF-9 was chosen as other published work hasindicated this concentration is optimal to induce mitogeniceffects. An additional 150 �L of 10% 1X HY medium wasadded to wells in one plate and served as the control (with-out gel, monolayer culture). Meanwhile, 150 �L of the K-63I

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polymer gel was added to wells in the other plate for a totalof 300 �L of the cell/media/polymer suspension in each ofthe remaining wells. The final cell/polymer suspension wascomprised of no more than 4–5% (of polymer) by weight in1X media. The contents of each well were mixed thoroughlywith a pipette to ensure that cells were evenly dispersed andsuspended in the control wells and in the polymer solution,respectively. The plates were incubated immediately for 1 hat 37°C in 5% CO2 to allow the polymer to solidify. Anadditional 150 �L of 10% 1X HY media containing heparin,with and without the FGF-9 (each adjusted for the correctFGF-9 final concentrations), was gently pipetted over thecontrol wells and the solidified gels. Care was taken toensure that the media did not dissolve the gelled matrix andthat the plate containing the gel remained at a temperatureabove 37°C at all times to prevent liquefaction of the poly-mer, keeping the cells immobilized in situ.

After 3 days in culture, harvesting of cells grown in 3D gelculture was facilitated by dissolving the polymer gel in eachwell with 1000 �L of cold HBSS and placing the plate atroom temperature for approximately 15 min to allow thepolymer to liquify. After removing the contents of each well,both monolayer and gel-containing plates were trypsinizedwith 0.25% trypsin to detach cells remaining on the plates.HBSS was used to dilute the trypsin and wash each well.Because of the cell retrieval technique requiring severalwashes, proliferation rates were only estimated indirectly bytotal cell yield. Aliquots of the cells retrieved were enumer-ated and assayed for cell viability using the trypan blueuptake method while another aliquot assessed phenotypeexpression at the mRNA level. The presence of chondrocyticmarkers including collagen type II and aggrecan were de-termined using reverse transcription polymerase chain reac-tion (RT-PCR) analysis.

Cells were enumerated according to the trypan blue up-take method. Using a hemacytometer, unstained cells wereconsidered viable while cells taking up the blue dye wereconsidered dead. Cells (1 � 104) were fixed with CytospinCollection Fluid (Shandon Inc.) and cytocentrifuged for 6min onto lysine-coated slides. The HEMA 3 Stain Set (Bio-chemical Sciences Inc.), which utilized a three-step stainingprocess, was used to stain the slides so they exhibited highcontrast and granular resolution. Slides were examined un-der light microscopy.

The TRIzol (Life Technologies, Rockville, MD) reagentmethod was used to isolate total RNA. Total cDNA librarieswere synthesized using 1 �g of total RNA, the AdvantageRT-PCR kit (Clontech Laboratories, Palo Alto, CA), and theOligo (dT18) primer. The SuperTaq Plus (Ambion, Austin,TX) PCR kit and specific primers for collagen types I and II,proteoglycans-aggrecan, and the housekeeping gene S14 ri-bosomal subunit expanded the resulting reverse transcrip-tase product.18 Each PCR reaction required the use of 20 mgof cDNA. Primers for collagen type I (sense, 5� GAC GGGAGT TTC TCC TCG GGG TC 3�; antisense,5� GAG TCTCCG GAT CAT CCA CGT C 3�), collagen type II (sense, 5�CAC CTT GGA CGC CAT GAA GGT 3�; antisense, 5� GTGAAC CTG CTA TTG CCC TCT 3�), and aggrecan (sense, 5�GGG TCA ACA GTG CCT ATC AG 3�; antisense, 5� GGGTGT AGC GTG TAG AGA TG 3�) were used for amplifica-tion. PCR reactions for collagen type I, collagen type II, andaggrecan were conducted in a PerkinElmer Thermal Cycler.

Thirty of the following cycles were performed: denaturation(94°C–15�, annealing (65°C—15�), and extension (68°C—3�)following initial treatment (75°C—5� and 94°C—1�). TheUN-SCAN-IT Gel Automated Digitizing System (Silk Scien-tific Corp.) was used for densitometry while agarose gelelectrophoresis was utilized to analyze the PCR products.

Data was analyzed using the STAT program with multi-ple comparison using one-way analysis of variance(ANOVA). Differences were considered statistically signifi-cant at p � 0.05.

RESULTS AND DISCUSSION

Dynamic rheometry methods were employed to in-vestigate the temperature-dependent properties of so-lutions and gels. At room temperature, aqueous solu-tions of all synthesized copolymers are able to flowfreely. However, at physiological temperatures thesol–gel transition results in soft gels. The gels formedare known as physical gels exhibiting gel–sol transi-tions allowing them to be fully reversible. The K-63Iwas chosen for use with the FGF-9 based on its highmolecular weight, appropriate stiffness, and ability togel and liquefy rapidly, making it efficient for cellseeding and retrieval. The K-63I polymer gel was non-toxic as cell viability and cell number were similar tomonolayer. Chondrocytes grown in monolayer cul-ture could be seen to adhere to the bottom of the platesalthough chondrocytes propagated in the polymerwere not visible under a light microscope. When in-cubated, the polymer transformed from a transparentliquid form to an opaque gelled form where the cellswere held in situ. Because the growth of chondrocytesin the gel was approximated to parallel the increase incell number of chondrocytes grown in monolayer, thegrowth of cells propagated in the polymer could notbe observed. Upon attachment to the bottom of theplate, chondrocytes in monolayer culture appearedstretched out and became densely packed followingseveral days in culture.

Photomicrographs of articular chondrocytesshowed cell morphology and whether the cells hadany mitogenic effects (Fig. 1). The cells retrieved frompolymer culture appear to be clumped together andstill retain their spherical shapes [Fig. 1(B,D,F, and H)].Cells from monolayer containing FGF-9 are roundwith well-defined cell membranes and not readily dis-tinguishable from cells grown in monolayer culturewithout the addition of FGF-9 [Fig. 1(C and G)]. Thereis little to no detectable difference between cells re-trieved from monolayer culture despite the addition ofFGF-9 during culture [Fig. 1(A,C,E, and G)]. Cellsretrieved from the monolayer cultures are typical ofcells grown in monolayer in that these cells appearspherical and refractile and exhibit distinct nuclei.Chondrocytes retrieved from the K-63I polymer that

FGF-9 AND CHONDROCYTE CULTURE IN THERMOREVERSIBLE POLYMERS 369

have also been treated with FGF-9 [Fig. 1(D and H)]display morphological properties similar to chondro-cytes taken from the K-63I polymer without FGF-9[Fig. 1(B and F)]. Cells from polymer culture [Fig.1(B,D,F, and H)] appear to adhere to one anotheralthough the cells themselves are still spherical. Cellspropagated in the polymer [Fig. 1(B,D,F, and H)] havefuzzy outlines and the boundaries of the cells are notas well defined as cells taken from monolayer culture[Fig. 1(A,C,E, and G)]. The ‘halo‘ around these cellsmay be attributed to the residual polymer remainingaround the cells that may not have washed off com-pletely during retrieval or to the possible synthesis ofan ECM by the cells. The difference in cell morphologycould only be detected between cells retrieved frommonolayer and 3D culture as the addition of FGF-9failed to induce a change in cell morphology.

Three different cell lines of chondrocytes isolatedfrom human articular cartilage were 80% viable inboth monolayer and 3D polymer cultures [Fig. 2(A,B)].However, the addition of FGF-9 to the two types ofculture did not significantly alter cell viability or cellnumber (p � 0.05). In contrast, the number of viablecells retrieved from the 3D culture (1.4 � 105) was upto twice the number of viable cells retrieved fromcontrol monolayer cultures (0.7 � 105) despite theaddition of FGF-9 [Fig. 3(A,B)]. Although the samenumber of cells were seeded in the polymer, the num-ber of retrieved cells varied according to the cell line.Line 3 showed higher cell yield with the addition ofFGF-9 in monolayer culture [Fig. 3(A), p � 0.05). How-ever, there is no significant difference in cell numberwhen FGF-9 is added to gel culture [Fig. 3(B)]. There

Figure 1. Photomicrograph of articular chondrocytes retrieved from FGF-9-containing cultures. Phase contrast photomi-crographs depicting the morphology of line 2 of human articular chondrocytes retrieved after 3 days in culture. Cells havebeen fixed onto lysine-coated slides by cytocentrifugation and were stained using the HEMA 3 Stain Set. The top row showscells at a magnification of 2500 while the bottom row shows cells at a magnification of 4000. (A, E) Control monolayer alone.(B, F) K-63 polymer gel alone. (C, G) Monolayer containing FGF-9. (D, H) K-63 polymer gel containing FGF-9. Chondrocytesin the monolayer culture are round with distinct nuclei while cells propagated in the K-63I polymer appear to have formedcell aggregates, although the cells remain spherical.

Figure 2. Effect of FGF-9 on the total number of chondro-cytes enumerated in polymer and monolayer cultures. Av-erage number of cells retrieved from different cell lines ofhuman articular chondrocytes seeded in (A) control mono-layer cultures and (B) 3D polymer cultures following 3 daysin culture. The white bars serve as the control, indicating thecells have been cultured without FGF-9. The dark bars indi-cate the number of cells retrieved from culture containingFGF-9.

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was thus a higher number of cells retrieved from 3Dculture than monolayer culture despite the presence ofthe FGF-9. Moreover, the addition of FGF-9 to bothtypes of cultures failed to alter the number of viablecells retrieved from each type of culture although thepolymer gel was determined not to be cytotoxic.Chondrocytes retrieved from control and 3D culturesin the presence of FGF-9 did not show upregulation ofcartilage-specific components, indicated by their RT-PCR profiles (Fig. 4). Cells propagated in the gel (Fig.4, columns 2 and 4) showed little to no decrease incollagen type I expression compared to cells propa-gated in monolayer culture. In contrast, there is en-hanced expression of collagen type II and aggrecan forcells seeded in the polymer (Fig. 4, columns 2 and 4).Equal loading of the wells was confirmed by similarintensities of the S14 housekeeping gene (Fig. 4, toprow).

Our previous study showed that the K-63I formu-lation of the poly(NiPA-co-AAc) thermoreversiblesupports cell growth, viability, and metabolic activity.16

This study confirms that chondrocytes grown in thepolymer gel proliferate faster then cells grown inmonolayer culture following 3 days in culture. Be-cause the K-63I formulation supports chondrocyte

propagation, we sought to determine whether addi-tion of the FGF-9 would further enhance chondrocyteproliferation and production of ECM components.FGF-9 has been shown to have a mitogenic effect onrat clonal chondrocytes.19 FGF-9 also induced ratclonal chondrocytes to progress through the differen-tiation pathway normally seen in chondrocytes. Therole of FGF-9 as a mitogen and differentiating factorfor developing tissues has only recently been studied.When treated with FGF-9, glial cell growth is stimu-lated.20 In addition, FGF-9 has been identified to playan integral part in development and in adult nervoussystems. However, limited studies have been con-ducted to determine the effect of FGF-9 on articularchondrocytes in vitro.

The key discovery in this study is that the FGF-9 didnot enhance chondrocyte multiplication. This findingwas not unexpected as our previous investigationshowed that the polymer alone provided an environ-ment that facilitated cell proliferation. There was also

Figure 3. Effect of FGF-9 on chondrocyte viability mea-sured by trypan blue uptake. Percentage of viable cellsretrieved from different cell lines of human articular chon-drocytes seeded in (A) monolayer and (B) 3D cultures usingK-63I polymer gels following 3 days in culture. The whitebars serve as the control, indicating the cells have beencultured in monolayer or in a scaffold without FGF-9. Thedark bars indicate the total percentage of viable cells re-trieved from culture containing FGF-9.

Figure 4. Expression of ECM components for chondrocytespropagated in FGF-9-containing cultures. The RT-PCR gelshows the effects on phenotype expression of human artic-ular chondrocytes retrieved from monolayer and gel-con-taining cultures with and without treatment with FGF-9. Inparticular, line 2 demonstrates the effect of the FGF-9 andK-63I polymer on the expression of collagen types I and II, aswell as aggrecan. Following 3 days in culture, the chondro-cytes are frozen at �70°C and RNA extraction was per-formed for RT-PCR analysis. The S14 housekeeping genewas used to ensure equal loading in the wells. The pheno-type expressions for (1) control monolayer without treat-ment, (2) K-63 polymer gel without treatment, (3) monolayertreated with FGF-9, and (4) K-63 polymer gel treated withFGF-9 are shown in the RT-PCR profile.

FGF-9 AND CHONDROCYTE CULTURE IN THERMOREVERSIBLE POLYMERS 371

continued production of ECM components such ascollagen type II and aggrecan, suggesting that thechondrocytes maintained features of their originalphenotype when seeded in the polymer.16 Hence, thethermoreversible polymer is able to create a scaffoldmimicking the extracellular matrix, enabling cells toproduce ECM components in the absence of FGF-9. Itis possible that the effects of FGF-9 could have beenmore pronounced at higher concentrations. The con-centration used (5 �g/mL) was found to be the mosteffective in inducing cell multiplication in previousstudies.21 Thus, the concentration chosen would elicitsimilar mitogenic effects on other culture systems un-less the cells have already reached their maximumproliferative response. It is likely that in the polymerculture chondrocytes are at their maximum prolifera-tive capacity. Lower concentrations of FGF-9 than thatchosen for use would not have elicited a response inthe chondrocytes while higher concentrations havenot been shown to further promote chondrocytegrowth in monolayer cultures. That the FGF-9 couldbe more effective at exposure times longer than 3 dayscould not be evaluated in the polymer gel system. The3-day incubation period also ensures that the FGF-9retains its biologic activity. Past 3 days in culture,overcrowding in the wells occurs and cells begin todie. Thus, longer incubation periods of chondrocytesin the polymer culture, with and without FGF-9, arenot feasible. This study confirms that the polymerculture system provides the optimal milieu to supportchondrocyte proliferation and function.

This work was supported by the Department of EnergyOffice of Biological and Environmental Research and theJohns Hopkins University School of Medicine and Depart-ment of Orthopaedic Surgery. A.A. is recipient of the 2002Johns Hopkins University Provost’s Undergraduate Re-search Award. Discarded nasal cartilage was supplied fromDr. Alan Shikani and the Good Samaritan Hospital. OliverPerez, Jinny Ha, and Shirley Anderson helped in the prep-aration of the article.

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