tracking chondrocytes and assessing their proliferation with pkh26: effects on secretion of...

Tracking Chondrocytes and Assessing Their Proliferation withPKH26: Effects on Secretion of Proteoglycan 4 (PRG4)

Kanika Chawla,1 Travis J. Klein,1 Barbara L. Schumacher,1 Tannin A. Schmidt,1 Michael S. Voegtline,1

Eugene J-M.A. Thonar,3-5 Koichi Masuda,3,4 Robert L. Sah1,2

1Department of Bioengineering, University of California–San Diego, 9500 Gilman Dr., MC 0412, La Jolla,California 92093-0412

2Whitaker Institute of Biomedical Engineering, University of California–San Diego, La Jolla, California

3Department of Biochemistry, Rush University Medical Center, Chicago, Illinois 60612

4Department of Orthopaedic Surgery, Rush University Medical Center, Chicago, Illinois 60612

5Internal Medicine, Rush University Medical Center, Chicago, Illinois 60612

Received 9 February 2005; accepted 31 October 2005

Published online 19 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20116

ABSTRACT: Distinguishing between implanted and host-derived cells, as well as between distinctcell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. Thefluorescent tracker dye, PKH26,was previously applied to several cell types to assess proliferation invitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 fortracking chondrocytes from superficial and middle zones and their proliferation, and determine theeffects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated ineither low- or high-densitymonolayer cultureandanalyzed for retentionofPKH26byflowcytometryand fluorescencemicroscopy at days 0 and 7. Cell suspensions and conditionedmedia were analyzedfor DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so thatthe number of cells in each doubling generation contributing to the final cell population could beestimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles ofdivision. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could bedistinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cellfluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis ofPRG4 suggest that PKH26 can be useful in determining the fate and function of implantedchondrocytes in vivo, as well as monitoring proliferation in vitro. � 2006 Orthopaedic Research

Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1499–1508, 2006

Keywords: PKH26; flow cytometry; proliferation; proteoglycan 4 (PRG4);chondrocyte

INTRODUCTION

Recent studies have examined a number of cell-based tissue-engineering methods for repairingfocal cartilage defects.1 Cells can be delivered intothe defect by direct injection,2 under a periosteumflap,3 within a fibrin delivery vehicle,4 or withinphotopolymerizable hydrogel.5 Alternatively, cellscan be delivered within scaffolds cultured for short(hours to days)6 or long (weeks to months)7,8

durations, as a cartilaginous construct synthe-sized without a scaffold9 or as a cartilaginous layeras part of a composite scaffold.10 Retention of theimplanted cells is generally assumed to facilitate

cartilage repair and regeneration in all theseprocedures. However, cell retention can be proble-matic2,6 and delivery by arthoscopic implantation,although having the advantage of being minimallyinvasive, presents additional challenges for cellretention.

Methods for tracking chondrogenic cells in vitroand in vivo vary in terms of tracer detectability atlonger follow-up and versatility in distinguishingcellular organization and cell populations. A vari-ety of probes can be applied. Most fluorescentcytoplasmic dyes (CellTrackerTM Green CMFDA,CellTrackerTM Orange CMTMR, BCECF-AM;Invitrogen, Carlsbad, CA) are limited to short-termstudies of only a few days11 due to short dye half-lives. In contrast, cell transduction with a reportergene such as b-galactosidase (lacZ)12 or GreenFluorescent Protein (GFP)13 allows for cell

JOURNAL OF ORTHOPAEDIC RESEARCH JULY 2006 1499

Correspondence to: Robert L. Sah (Telephone: 858-534-0821;Fax: 858-822-1614; E-mail: [email protected])

� 2006 Orthopaedic Research Society. Published by Wiley Periodicals,Inc.

tracking as long as the gene is retained andexpressed. However, transfection efficiencies ofgene delivery into cells, especially in primaryisolates or early passages, can be technicallydifficult, and result in transient expression;14

infection with viral vectors is also challenging,and can invoke an immune response.15 Radioactiveprobes, such as tritiated thymidine16 and thethymidine analog bromodeoxyuridine (BrdU),17

can also be useful for longer term cell tracking,depending on probe lifetimes. Newly synthesizedDNA has been detected after as long as 12 weeks invivo.17 However, determining organization of radi-olabeled cells is difficult, and quantitative analysisis limited to two-dimensional histological sectionsby autoradiography.18 Alternatively, cell popula-tions can be distinguished by intrinsic cell proper-ties. The testis sex-determining region Y gene,SRY, which is present in cells originating frommale but not female donors or recipients, has beenused in primary and expanded cells and has beenvalidated in studies up to 12weeks.6 This limits celltracking to allogeneic, gender-specific situations.6

For all of the above methods, the division history ofthe implanted cells is difficult to assess. Thus,tracking chondrocytes with a more stable fluores-cent dye would be useful.

Apreviously developed stable family of lipophilicfluorescent dyes has been used for cell trackingtransplanted cells. PKH dyes19 are stably incorpo-rated into the cell membrane and appear to benontoxic.20–22 One of these dyes, PKH26, has beenused to track various cell types.23,24 BecausePKH26 labels the membrane, similar to lipophilicdyes DiI and DiI derivatives,11,25 the associatedfluorescence signal decreases by half with each celldivision cycle.26 This characteristic has been usedto determine the cell number in each doublinggeneration contributing to a final cell population,thereby assessing proliferation.20,27–29

Tracking chondrocytes with PKH26 could beuseful for analysis of cartilage repair in vivo.However, the effects of PKH26 on chondrocytefunctions should first be assessed. In repair andin homeostasis, proliferation and secretion of thefunctional marker molecule proteoglycan 4(PRG4), encoded by the PRG4 gene,32 and alsotermed Lubricin,30 or Superficial Zone Protein31

are important. Cartilaginous constructs can befabricated in a stratified form so that PRG4secretion is localized at the tissue surface.34 Thus,the objectives of this study were to establish a cell-tracking method using PKH26 for generationanalysis of chondrocytes from superficial anddeeper zones and to determine the effects of

PKH26 on proliferation and secretion of PRG4protein.

MATERIALS AND METHODS

Materials for cartilage explant, chondrocyte isolation,monolayer culture, and biochemical procedures wereobtained as described previously.16,34 In addition, bovineserum albumin (Fraction V, cell culture tested) andPKH26 labeling kits were obtained from Sigma (St.Louis, MO). Tissue culture treated flasks were fromCorning (Corning, NY). Trypsin-EDTA and Hanks’Balanced Salt Solution (HBSS) were from Invitrogen-Gibco (Grand Island, NY).

Chondrocytes from the superficial and middle layersof immature bovine cartilagewere isolated.16,34Articularcartilage slices from the superficial (<0.2 mmdepth) andmiddle (0.4–1.0 mm) layers were harvested from thepatellofemoral groove of seven 1–3-week-old calf kneejoints and digested in medium (Dulbecco’s ModifiedEagle Medium [DMEM], 10 mM HEPES, 0.1 mMnonessential amino acids, 0.4 mM L-proline, 2 mML-glutamine, 100 U/mL penicillin, 100 mg/mL strepto-mycin, and 0.25 mg/mL amphotericin B) with 0.2%pronase for 1 h, and 0.02% collagenase-P for 16 h.

Superficial and middle zone chondrocytes wereexpanded in monolayer culture. Cells were plated at lowdensity (10,000 cells/cm2) and grown in tissue culture-treated175 cm2flasks in completemediumwith10%fetalbovine serum (FBS) and 25 mg/mLascorbic acid at 378C inan atmosphere of 5% CO2/95% air. Each flask received30mLmedium per 3 days until cells were 80% confluent.Cultures were then serum starved (incubated inmediumwith 0.01% BSA) for 1 day to synchronize cells. Uponrelease with 0.25% trypsin-1 mM EDTA in HBSS cellswere pelleted, counted, and resuspended in completemedium at about 1 million cells/mL.

To determine the extent of PKH26 retention and theeffects of PKH26 labeling on proliferation and secretionof PRG4 by superficial and middle chondrocytes, somecells were labeled with PKH26 (and subsequentlydesignated as S* and M*, respectively).

Some of the released chondrocytes (about 10 millionsuperficial or middle zone cells) were washed once inmedium without serum, pelleted, and resuspendedin 1 mL of Diluent C (dilution buffer provided inPKH26 cell labeling kit) according to manufacturer’sdirections. The cell suspension was mixed with an equalvolume of labeling solution containing PKH26 withdilution buffer (final concentration 20 mM).35 Chondro-cytes were incubated with the labeling solution for 5 minat 258C with periodic tapping of the tube. The stainingreaction was stopped by adding an equal volume of FBSand incubating for 1min. Cells werewashed and pelletedtwice thereafter, each time with 5 mL complete medium.

Different extents of cell division were examined byusing different seeding densities. Cells were analyzedimmediately or replated in 12- or 24-well dishes atdensities of 10,000 or 200,000 cells/cm2 and cultured in

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monolayer for an additional 7 days with 2 mL completemedium per million cells that was changed every 2 days.Cultured cells were then released with trypsin-EDTA(Fig. 1).

Qualitative assessments of cellular retention andeffects of the PKH26 tag were made by fluorescencemicroscopy (Nikon Eclipse TE 300, AG Heinze, Irvine,CA) with digital imaging (SPOT RT, Diagnostic Instru-ments, Burlingame, CA). Phase and fluorescent imageswere taken of the same field of view of labeled cells, andphase images were taken of untagged cells.

Samples of suspended cells (about 200,000–800,000cells/mL) were analyzed by flow cytometry. Retention ofPKH26 and proliferation by superficial and middle zonechondrocytes were assessed from dye fluorescence inten-sity profiles. A FACScan flow cytometer with argon ionexcitation laser at 488 nm (Becton-Dickinson, FranklinLakes, NJ; voltage to the fluorescence detector¼ 395 V)was used to analyze samples. Fluorescence representingPKH26 (peak excitation at 551 nm, peak emission at567 nm) was collected through a 585/42 nm filter. Withthese settings, fluorescence intensity values spanned4 log decades or 1024 channels on a linear scale. For eachsample, data representing 10,000 cells were collected asa list-mode file using CellQuestTM software (BectonDickinson) and then analyzed using FlowJoTM software(Tree Star, Inc., version 5.3, Ashland, OR). Nonviablecells were identified based on propidium iodide staining(data not shown), which had higher signal than PKH26,lower forward scatter intensity, and higher side scatterintensity, and not analyzed further.20,27–29 A 5% thresh-old value was used as a gate for discriminating betweenuntagged control cells and tagged cells.

Fluorescence decay associated with PKH26 wascalculated assuming that decay obeyed first-order expo-nential kinetics.36 The decay was subtracted fromfluorescence associated with cells in generation 1. Eachsubsequent generation thereafter was assigned half thefluorescence of the previous generation, equivalent to aloss of 19.18 channels per generation. Then, a prolifera-tion index (PI) was calculated based on areas of eachmodel-generated distribution contributing to the samplehistogram.29 PI, a measure of proliferation, was definedas the ratio of the total number of cells (summation of theGaussian areas in each generation, Ak up to k¼ 10generations) to the calculated number of cells initiallyresiding in the parent generation (progenitor cells seededat day 0):

PI ¼

P10

k¼1

Ak

P10

k¼1

Ak

2k�1

ð1Þ

An index of net fluorescence, taking into accountproliferation, was calculated as the PI and the meanfluorescence of the sample.

Numbers of cells per generation (cycle of cell division)were determined by fitting day 7 histograms with aGaussian model of fluorescence distribution, based onday 0 data.29 A custom program was written in Excel(Microsoft 2002) to decompose sample histograms intoa sum of distributions representing successive genera-tions by nonlinear least-squares fitting. Thus, assum-ing that fluorescence halved with each cell divisioncycle, the number of cells in each doubling generation

Figure 1. Schematic of methods used to evaluate PKH26 retention by chondrocytesand effects of PKH26 on cell growth and PRG4 secretion. Cells were isolated separatelyfrom superficial and middle zones of articular cartilage and allowed to expand inlow density monolayer culture. After release, some cells were labeled with PKH26(indicated by *). Cells were either analyzed immediately or replated in monolayer at low(10,000 cells/cm2) and high (200,000 cells/cm2) densities for an additional 7 days and thenreleased. Superficial (gray) and middle (white) zone chondrocytes are shaded differentlyto emphasize their location in culture.

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that contributed to the final cell population was deter-mined.

Cells cultured inmonolayer at low and high densities,with and without PKH26, were solubilized with protei-nase-K.34 Portions of the digest were analyzed for DNAusing PicoGreen137 with calf thymus DNA as thestandard. Cell proliferation was quantified as DNAcontent relative to the number of cells initially seeded,assuming 7.8 pg DNA/cell.38

Spent media were analyzed for PRG4 secretion bychondrocytes from both zones by indirect ELISA usingmonoclonal antibody 3A4 (courtesy B. Caterson, Wales,UK).34 PRG4 levels were calculated from a standardcurve generated from samples containing knownamounts of PRG4 based on protein content.31 Amountsof PRG4 secreted were expressed as pg/(cell �day).

The effects of tagging, seeding density, cell type,day (for some parameters), and interactions wereassessed by ANOVA and post hoc Tukey tests, a¼ 0.05(Systat Software 10.2, Richmond, CA). Fluorescence,proliferation, and index of net fluorescence were logtransformed to improve the uniformity of varianceamong the experimental groups. Data were expressedas mean�SEM.

RESULTS

Phase and fluorescence images of unlabeled super-ficial and middle and PKH26-labeled (S*, M*)chondrocytes indicated that PKH26 labeled essen-tially all cells, and that the initial plating densityaffected the fluorescence intensity present after7 days of culture. Unlabeled cells plated at low andhigh densities did not exhibit any fluorescence,indicative of PKH26 labeling, as expected(Fig. 2A–D). By day 7, many of the PKH26-labeledcells that were seeded at low density (Fig. 2Eand G) appeared dimmer than cells initially seededat high density (Fig. 2F and H) but were still visi-ble. Comparison of phase (Fig. 2I–L) and fluores-cence (Fig. 2E–H) micrographs of PKH26-labeledcells indicated cells retained the dye effectively.

A high proportion of labeled cells retainedPKH26-associated fluorescence after 7 days ofculture, as analyzed by flow cytometry. Bothsuperficial and middle zone cells were labeledbrightly and with a high efficiency at day 0(98.9� 0.3% and 97.9� 1.1%, respectively, Fig. 3).By day 7, 97.3� 0.4% of superficial cells seeded atboth densities were fluorescently tagged, as weremiddle zone cells seeded at high density (Fig. 3).Middle zone cells seeded at low density proliferatedmore, so a lower percentage (87.9� 5.3%) retainedsufficient PKH26 to be identified after 7 days.

Generation analysis of flow cytometric histo-grams (Fig. 3) allowed discrimination of cells up

through at least 7 successive generations (based onthe location of a 5% gate, identifying unlabeledcells). A representative day 0 histogram shows thefluorescence distribution of cells immediately afterlabeling with PKH26 (Fig. 4A) with the calculateddistribution of cells per generation (Fig. 4B). Asexpected, the majority (93% for the sample inFig. 4B) of the cells were computed to be in the firstgeneration. After 7 days, some cells had diminishedintensity. A shift in the fluorescence distribution ofcells by day 7 was observed (Fig. 4C), with cellsseededat lowdensity shifted themost.Compared tothe day 0 distribution (Fig. 4B), the distribution ofcells per generation for these cells (Fig. 4D) wasnoticeably wider, with cells distributed amongmany generations; the largest percentage (32%) ofcells was in generation 5.

According to generation analysis, cell prolifera-tionwas affected by seeding density (p< 0.001) butnot chondrocyte origin (p¼ 0.33), although a sig-nificant interaction of the two factors (p< 0.05)wasfound (Fig. 5). At day 0, 80.2� 2.3% of the cells ineach populationwere calculated to be in generation1 (Fig. 5A and D). As expected, at day 7, more thanhalf of the cells seeded at high density resided ingeneration 4 or lower, compared to cells seeded atlow density, which had a high percentage of cellsshifted into higher generations (Fig. 5B and C).Although seeding density had an overall effect, themedian generation for superficial cells seededat low density was 4.9� 0.3, significantly higher(p< 0.001) than the median generation of 2.3� 0.4at high cell density (Fig. 5E and F). No significantdifference was found between the median genera-tion of middle zone cells seeded at low or high celldensities (p¼ 0.17).

The PI calculated from the generation model forS* andM* chondrocytes after 7 days of culture wassignificantly affected by cell type ( p< 0.05) anddensity (p< 0.001) with a significant interaction ofthe two factors (p< 0.05). The PI was greater(p< 0.001) in superficial cultures initiated at lowdensity (7.2� 0.7-fold) than those initiated at highdensity (2.7� 0.7-fold). Middle cultures seeded atlow and high densities proliferated at approxi-mately the same rates ( p¼ 0.6) (7.9� 0.1-fold, low;5.9� 0.9-fold, high) (Fig. 6A). By day 7, the numberof middle zone cells present at high density was3.1� 0.9 times more than superficial cells at highdensity (p< 0.05). At low density, the PI was notdifferent (p¼ 1.00) between cell types (Fig. 6A).

The mean fluorescence intensity of PKH26-labeled cells after 7 days was significantly affectedby density (p< 0.001) and cell type (p< 0.001)witha trend towards an interactive effect (p¼ 0.08;

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Fig. 6B). Intensity of cells seeded at low densitydecreased 12.9� 2.6-fold after 7 days (p< 0.001)while those seeded at high density decreasedsignificantly less, 6.5� 1.0-fold (p< 0.001). Meanintensity of superficial cells was 2.5� 0.4-fold(p< 0.001) higher than middle zone cells after

7 days. Intensity of cells seeded at high density was2.5� 0.3-fold (p< 0.05) higher than cells seeded atlow density by day 7.

The index of net fluorescence was significantlyaffected by cell type (p< 0.001) and density(p< 0.001), with a significant interactive effect

Figure 2. Effects of cell growth and seeding density on PKH26 retention by PKH26-labeled S* and M* chondrocytes. Phase (E–H) and fluorescence (I–L) micrographs of S*andM* cells inmonolayer culture at day 7 are shown.Micrographs of S* andM* cells areof the samefield of view.UnlabeledS (A,B) andM (C,D) cells are also shown.Cells seededat low (10,000 cells/cm2; A, C, E, G, I, K) and high (200,000 cells/cm2; B, D, F, H, J, L)seeding densities are indicated. Bar¼ 100 mm. [Color scheme can be viewed in the onlineissue, which is available at http://www.interscience.wiley.com]



(p< 0.05, Fig. 6C). By day 7, superficial chondro-cytes seededathighdensityhada2.4� 0.2-fold lossin the index ( p< 0.001) while middle zone cellsseeded at low density had a 2.8� 0.1-fold loss(p< 0.001). At low density, superficial cells had a2.5� 0.4-fold greater loss in the index compared tomiddle zone cells at lowdensity (p< 0.001). Cells athighdensity alsohadahigher loss in the index thancells seeded at low density (2.1� 0.5-fold super-ficial; 2.7� 0.3-fold middle) (p< 0.05). Thus, whenproliferation was considered, the net fluorescence(associated with the entire population of cells in agiven culture condition) was altered 2.0� 0.2-fold,on average, during in vitro culture.

Cell proliferation over the 7 days remainedunaffected by PKH26 labeling (p¼ 0.33), but was

significantly affected by density (p< 0.001) with atrend for an effect of cell type on relative prolifera-tion (p¼ 0.08). No significant interactions werefound (p¼ 0.12–0.98). During the 7 days, prolif-eration was 1.3� 0.2-fold greater by middle zonethan superficial cultures (p¼ 0.08) and greater(p< 0.001) in cultures initiated at low (9.7� 1.0-fold, superficial; 13.2� 1.2-fold, middle) than thoseinitiated at high density (2.0� 0.1-fold, superficial;2.2� 0.2-fold, middle) (Fig. 7A).

PRG4 secretion by superficial cells was signifi-cantly greater than that by middle zone cells(p< 0.001) and significantly affected by density(p< 0.001) but not by PKH26 tagging (p¼ 0.81)(Fig. 7B). A significant interactive effect occurredbetween cell type and density (p< 0.001). Super-ficial chondrocytes at low density secreted 1.1�0.3 pg/(cell �day) by day 7 in culture, significantlylower than the 9.0� 1.6 pg/(cell �day) secreted bysuperficial cells seeded at high density (p< 0.001).Middle zone cells secreted PRG4 at a much lowerrate, averaging 0.3� 0.1 pg/(cell �day), than super-ficial cells at high density (p< 0.001), but this ratewas not significantly different from superficial cellsseeded at low density (p¼ 0.76–0.92).

Figure 3. Effects of seeding density and time inmonolayer culture on PKH26 retention by (A) S* and(B) M* chondrocytes. Cell cycles were synchronized by1 day of serumstarvation prior to release frommonolayerexpansion culture. Cell suspensions were analyzed forfluorescence by flow cytometry. Percentage of cellsretaining PKH26 were calculated for low and high celldensities based on the position of gate for unlabeled cells(dotted line) (n¼ 6–7).

Figure 4. Cell suspensions were analyzed for fluores-cence by flow cytometry. Numbers of cells per generation(cycle of cell division) were determined by fitting day7 histograms from a model of Gaussian fluorescencedistribution, based on day 0 data. (A) Representativesample of labeled cells on day 0. (B) Accompanyingdistribution of cells per generation for the day 0 sampleshown in (A). (C) A representative sample of superficialcells initially seeded at 10,000 cells/cm2 at day 7 withrespective generation numbers indicated. (D) Accompa-nying distribution of cells per generation for the sampleshown in (C).

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DISCUSSION

These results establish methods for using PKH26in studies of chondrocyte growth without adverseeffects on chondrocyte function, specifically, cellu-lar proliferation and secretion of PRG4. Fluores-cence microscopy (Fig. 2) and analysis of cellsuspensions by flow cytometry (Fig. 3) bothindicated strong retention of PKH26 by superficialand middle zone chondrocytes after 7 days inmonolayer culture, whether cells were induced toproliferate at a low rate (seeding at high density,200,000 cells/cm2) or at a high rate (seeding at alow density, 10,000 cells/cm2). By day 7, fluores-cence in 91% of these cells was still detectableby flow cytometry, while the net fluorescencewas somewhat decreased for both cell types andseeding densities (Fig. 6). The possibility of

tracking PKH26-labeled cells through severalgenerations was established from flow cytometricdata (Figs. 4 and 5). PKH26 labeling did not affectproliferation rate or secretion of PRG4 (Fig. 7).

Figure 5. Effects of chondrocyte origin and seedingdensity on proliferation. Chondrocytes were isolatedfrom the superficial and middle layers of articularcartilage, plated at low or high cell densities, culturedfor 7 days, and analyzed for the percentage of cells indifferent generations. (A) Distribution of superficial (^)and middle (}) chondrocytes per generation at day 0. Atday 7, superficial (B) andmiddle (C) chondrocytes platedat low (&) and high (~) densities were analyzed for thepercentage of cells in different generations. Mediangenerations were also determined (D–F) [n¼ 6–7;p¼ 0.001 (*)].

Figure 6. Effects of chondrocyte origin and seedingdensity on retention of PKH26 in vitro. (A) Proliferationindex of tagged cells at day 7, normalized to proliferationindex of day 0 samples, as determined from generationmodel. (B) Mean fluorescence of cells at day 0 and day 7.(C) Index of net fluorescence was calculated as product ofmean fluorescence and proliferation index [(n¼ 6–7;p< 0.001 (*), p< 0.05 (^)].



In the future, longer culture durations and othercell sources should be analyzed, because such cellcultures may exhibit different responses. In addi-tion, the effects of PKH26 on a variety of cellfunctions should be delineated. Although prolifera-tion is an important feature of isolated chondro-cytes for tissue engineering, and PRG4 expressionis characteristic of superficial cells, cell fateprocesses such as (re)differentiation are alsoimportant and may be affected by PKH26.

PKH26 effectively labeled several celltypes22,24,39 in studies for tracking of lymphocytesduring 60-day homing studies in rats40 and germcells over 12weeks after transplantation in goats.41

PKH26 was also used to track the fate of four-cellstage bovine embryos during blastocyst forma-tion.42 Localization of PKH26-labeled cells is usefulbecause the dye does not passively transfer to

unlabeled cells, such as after injection of labeledcells in vivo40 or in cocultures of labeled andunlabeled cells in vitro.24,40 PKH26 labelingwould seem applicable in cartilage repair studies,because, superficial and middle zone chondrocytesisolated from cartilage of 4–6-month-old Yucatanminipigs have also been effectively labeled,43 andPKH26-labeled chondrocytes injected in an auto-logous chondrocyte implantation procedure ingoats indicated retention of fluorescently labeledcells after 14 weeks in vivo.35

The utility of PKH26 for assessing cell prolifera-tion follows from its bright fluorescence and utilityfor cell tracking. Previous studies developed flowcytometricmethods for assessingproliferationwithPKH26 in cell types other than those that areskeletally differentiated; the reduction of fluores-cence intensity with chondrocyte division (Fig. 3)generally agreeswith such studies.20,27–29,44,45 Thediminution of fluorescence is consistent with dilu-tion during proliferation because PKH26-asso-ciated fluorescence does not decrease significantlyin nondividing cells.27,44

PKH26 does not adversely affect intrinsic cel-lular functions including proliferation (Fig. 7A),consistent with other studies.28,44,45 Effects onproliferation were similar, whether determinedwith PKH26 (by generation analysis) or by Pico-Green1 assay (for DNA content). Differences in theextent of proliferation, for example, with middlezone chondrocytes seeded at high density havinga higher proliferation index by generation analysisflow cytometry than byDNA assay, may be due to anumber of factors. Only viable cells were consid-ered during the flow cytometry analysis, whereasboth viable and nonviable cells were quantified byDNA content analysis. Also, photobleaching,expected with light exposure of cultures duringmedium changes, would lead to overestimation ofproliferation by generation analysis.

Ideally, supravital dyes should not have harmfuleffects on other cellular functions, such as PRG4secretion by chondrocytes. The inertness of PKH26labeling on PRG4 secretion by chondrocytes isconsistent with its lack of adverse effects on cellviability20–22 and growth28,40,44 and embryonicdevelopment.42 In the present study, PRG4 secre-tion levels were comparable to those found pre-viously for superficial and middle zonechondrocytes in monolayer culture.46

Tracking chondrocytes labeled with PKH26could have widespread applications for evaluatingimplanted cells in articular cartilage repair invivo, especially in biopsies or terminal analysesin animal models. Tracking culture-expanded

Figure 7. Effects of cell type, tagging, and cell densityon (A) relative proliferation and (B) PRG4 secretion.Relative proliferation was determined as the number ofcells present on day 7 (PicoGreen1 assay for DNA)normalized to number of cells seeded. Spent mediumcollected on day 6 of culture was analyzed for PRG4secretion by indirect ELISA using monoclonal antibody3A4 andnormalized to number of cells present on day 7 ofmonolayer culture (n¼ 4–7).

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chondrocytes could help determine cell retention atthe repair site and redifferentiation. Ideally, cellsfor implantation would be labeled as close aspossible to the implantation time to minimize thefluorescence decrease due to division prior toimplantation. Prior to applying PKH26 labeling totrack chondrocytes quantitatively, the number ofcell divisions that PKH26-labeled chondrocytescould undergo and still be detected had to bedetermined.Our results suggest that for constructsimplanted in vivo with PKH26-labeled cells, anyrecovered cells that were PKH26 positive wouldhave undergone less than seven cycles of cell divi-sion. Conversely, those that are PKH26-negativewould either be from the host or be implanted cellsthat have proliferated extensively.

Several lines of reasoning suggest that PKH26will be useful even in the presence of a localinflammatory joint reaction. The structure ofPKH2611 makes it unlikely to be cleaved byproteases, including collagenases, or other mam-malian enzymes (personal communication, Mole-cular Probes). Also, when PKH26-labeled cells arereleased with cell dissociation enzyme prepara-tions (e.g., based on trypsin), a high proportion(99%) is clearly labeled. The finding that PKH26labeling did not adversely affect chondrocyte pro-liferation and PRG4 secretion is significant forfuture in vivo cell tracking studies aimed at gaininginsight into mechanisms underlying articularcartilage repair.

ACKNOWLEDGMENTS

This work was supported by grants from NASA, NIH,NSF, and a predoctoral fellowship from the WhitakerFoundation (KC).

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