Bioreactor and Probe System for Magnetic ResonanceMicroimaging and Spectroscopy of Chondrocytesand Neocartilage

Erik Petersen,1 Kimberlee Potter,2 John Butler,1 Kenneth W. Fishbein,2 Walter Horton,3

Richard G. S. Spencer,2 Eric W. McFarland1

1 Department of Chemical Engineering, University of California, Santa Barbara, CA 93106-5080

2 Nuclear Magnetic Resonance Unit, National Institute on Aging, National Institutes of Health,Baltimore, MD 21224

3 Laboratory of Biological Chemistry, National Institute on Aging, National Institutes of Health,Baltimore, MD 21224

Received 30 October 1996; revised February 1997

ABSTRACT: We have developed a nuclear magnetic resonance which are presently being used clinically, may be greatly aided by(NMR)-compatible hollow fiber chondrocyte bioreactor (HFBR), per- in vitro studies of chondrocyte growth in well-controlled systems.mitting the noninvasive study of neocartilage under conditions opti- Because they are noninvasive, nuclear magnetic resonancemized for cell growth and matrix expression. The system was used (NMR) imaging and spectroscopy techniques have the potentialto investigate the properties of neocartilage which developed from to permit serial studies of morphology and metabolism duringembryonic chick chondrocytes. Histologic studies performed 30 days

chondrocyte growth and matrix production. Accordingly, weafter inoculation of the HFBR with chondrocytes showed cartilagepresent here the initial description of a model system for serialgrowth units demarcated by stromal layers surrounding each fiber;NMR studies of cartilage formation from chondrocytes. It is basedthe tissue itself was highly cellular with abundant proteoglycan con-on a hollow fiber bioreactor (HFBR) design in which the cellstent. Spin–density, spin–lattice, and spin–spin relaxation and magne-

tization transfer contrast images revealed heterogeneous tissue with are maintained at high cell densities [5] as required to preserveNMR properties that correlated well with histologic data. It was found the chondrocyte phenotype [4] , and for sufficient time for extra-that the apparent free water content of the neocartilage was greater cellular matrix to be generated.than that seen in mature cartilage, even in regions of relatively low A variety of NMR microimaging techniques [2] and contrastcell density. The bioenergetic profile of the cells in culture was moni- modalities were used to explore specific characteristics of chon-tored with 31P-NMR spectroscopy, and the presence of phosphocre- drocyte matrix production. The NMR images were correlatedatine was clearly demonstrated. Overall metabolic stability was con-

with histologic sections to provide a basis for interpretation offirmed between days 10 and 17 after inoculation. A significant de-the image features.crease in intracellular pH with time was observed during early

31P-NMR spectroscopy was used to identify the phosphorus-development of the chondrocyte system. q 1997 John Wiley & Sons, Inc.containing metabolites present at significant concentrations inInt J Imaging Syst Technol, 8, 285–292, 1997chondrocytes. In addition, 31P-NMR enabled us to monitor theviability and stability of chondrocytes in the reactor system. Bio-

I. INTRODUCTION chemical assays related to bioenergetics were performed in paral-lel with the NMR measurements.The prevalence and clinical importance of cartilage disease pro-

vides strong motivation for the study of chondrocyte develop- The work presented here shows the power of applying nonin-vasive magnetic resonance methods to neocartilage formation.ment. Attenuation of pathologic processes and implementation

of restorative procedures may best be examined by direct observa- These techniques clearly have great potential for investigatingthe effects of growth factors and inhibitors on chondrocyte devel-tion of chondrocyte proliferation and matrix production, rather

than with studies on whole intact cartilage. Indeed, further devel- opment and matrix production.opment of the techniques of in vitro human cartilage transplants,

II. EXPERIMENTAL METHODSA. Tissue Culture Medium. Tissue culture medium (TCM)

Correspondence to: E. W. McFarland was prepared by adding 50 mL of heat-inactivated fetal bovineContract grant sponsor: NIH; Contract grant number: R29-GM48887-04Contract grant sponsor: NSF; Contract grant number: DIR 91-96193 serum (Biofluids, Rockville, MD), 10 mL of 200 mM L-gluta-

q 1997 John Wiley & Sons, Inc. CCC 0899–9457/97/030285-08

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mixture of 95% air and 5% CO2 . To confirm integrity and sterilityof the system, incubation proceeded for 2 days prior to inoculationof the bioreactor with the cultured cells.

C. Reactor Inoculation with Chondrocytes. Chick chondro-cytes were isolated by collagenase digestion of sterna from day16 chick embryos using a modification of the protocols of Hortonet al. [8] and Gerstenfeld et al. [4]. Briefly, embryos were decapi-tated and the entire breast plate was excised. The sterna weredissected free of noncartilaginous tissue, cut below the ventralnotch, and placed in phosphate-buffered saline (PBS) (0.15 MNaCl). The sterna were then predigested in 5 mL of a 4-mg/mL collagenase (Worthington Biochemical Corp., Freehold, NJ)solution in PBS for 20 min. Then, to produce a single-cell suspen-sion, sterna were digested in 30 mL of collagenase solution at377C for 3 h with agitation. After digestion, the sample wascentrifuged for 3 min at 660 1 g , the supernatant was aspirated,and the cells were resuspended in TCM. The cell count wasdetermined using a hemocytometer. A typical yield from sixdozen sterna was 3.5 1 10 8 cells. Reactors were inoculated with107 cells using a syringe inserted through the rubber septum onthe side port.

Perfusion of the reactor was delayed by 4–8 h to facilitateadhesion of the cells to the fibers. After that time, flow was started

Figure 1. Schematic of NMR-compatible hollow fiber bioreactor at a rate of approximately 5 mL/min and was gradually increased(HFBR) system. The bioreactor is perfused by a peristaltic pump as

over the next 10 days to a rate of 14 mL/min.shown. The system is maintained at 377C and perfused continuouslywhile in the magnet.

D. Biochemical and Histochemical Analyses. Samples ofthe medium collected before every medium change underwentspectrophotometric analyses of glucose and lactate. All ab-

mine (Biofluids) , 1 mL of 250 mg/mL fungizone (Biofluids), sorbance measurements were made on a 96-well BioRad (Modeland 1 mL of 10 mg/mL gentamicin reagent solution (Gibco, 3550) microplate reader using 200 mL of a premixed solutionGaithersberg, MD) to a 500-mL bottle of Dulbecco’s modified incubated at room temperature for 5 min. For the glucose assay,Eagle’s medium with phenol red (DMEM; Biofluids). The me- 10 mL of medium was added to 1 mL of glucose reagent (Stan-dium was filter sterilized through a 0.2-mm cellulose filter. bio) . For the lactate assay, 10 mL of medium was added to 1 mLAscorbic acid (Sigma, St. Louis, MO) was added to the TCM of lactate reagent (Sigma Diagnostics). Absorbance readings forto a final concentration of 10 mg/mL at each change of medium lactate and glucose assays were made at 540 and 490 nm, respec-(i.e., at 48-h intervals). tively, and all readings were referenced to a calibration curve.

After imaging, the fibers and neocartilage were removed intactfrom the bioreactor and fixed in neutral buffered formalinB. Bioreactor. The bioreactors (Fig. 1) were constructed from

high-purity silicon glass tubing (o.d. 5 mm, height 60 mm). (Sigma) for 2 days at 47C. The tissue was then washed withPBS, dehydrated in a graded series of ethanol solutions, preequili-Inside each reactor were six porous polypropylene hollow fibers

(i.d. 330 mm, pore diameter 0.2 mm, wall thickness 150 mm; brated in xylene, embedded in paraffin (Paraplast ) , and sec-tioned perpendicular to the fiber axis. A 5-mm slice of tissueMicrogon, Inc., Laguna Hills, CA) potted with biomedical grade

silicon rubber (MED-1137; NuSil Silicone Technology, Carpen- was mounted on a microscope slide, deparaffinized, and thenstained with Alcian blue [13] , a metachromatic dye that bindsteria, CA). Reactors were flushed with 100% ethanol to enhance

the wettability of the fibers and then sterilized by soaking in a to the chondroitin sulfate side chains of the aggrecan proteo-glycan. All histologic slides were prepared by Paragon Biotech0.55% chloroform solution (Malinckrodt, Paris, KY) for at least

8 h. (Baltimore, MD) .The bioreactor circuit consisted of the bioreactor, a tempera-

ture-controlled 100-mL reservoir bottle and cap, a peristaltic E. Proton NMR Microimaging. All NMR experiments wereperformed with a Bruker AMX spectrometer coupled to a superpump, and 10 ft of gas permeable silicon tubing (size 14; Mas-

terflex). Unidirectional flow of the TCM was achieved by one- wide-bore magnet (diam. 104.7 mm) operating at 9.4 T (400.1MHz for 1H, 161.9 MHz for 31P). Proton NMR microimagingway valves located on either side of the pump interface. Before

connecting to a sterile bioreactor, the reservoir, tubing and valves was performed using a Bruker microimaging gradient probe witha 20-mm diameter resonator coil. The maximum gradient strengthwere filled with water and autoclaved for 30 min. After the biore-

actor was inserted into the loop, it was flushed with ethanol and used was 8 G/cm. The temperature of the probe was maintainedat 377C by a stream of heated air, and was monitored by a thermo-the water-filled reservoir was replaced with a reservoir of TCM.

Once assembled, the system was connected to a pin compression couple in close proximity to the bioreactor. TCM was also main-tained at 377C.pump (Cell Max Quad; Cellco Inc., Germantown, MD) and per-

fused with a fresh supply of TCM in an incubator gassed with a Several imaging contrast modalities were implemented to ob-

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tain quantitative maps of NMR-measurable parameters. The con-trast modalities used were: spin density (r ) , longitudinal (T1 ) andtransverse (T2 ) relaxation times, magnetization transfer contrast(MTC), from which a rate (km ) was derived to describe theinteraction of free and bound water, and longitudinal relaxationtime in the rotating frame (T1r) . Each image was perpendicularto the long axis of the reactor, showing the fibers in cross section,and had a field of view (FOV) Å 15 mm, slice thickness of 2mm, and matrix size of 256 1 256. Hence, the nominal in-planeresolution was 60 mm.

The theoretical signal intensity of a simple spin-echo imagingsequence with echo-time (TE) and repeat time (TR) is

I É r exp(0TE/T2 )[1 0 exp(0TR/T1 )] . (1)

A T1 map was obtained by fitting the signal intensity of 16 imagesobtained with TRs ranging from 0.1 to 10.0 s, with TE fixed at13 ms, to the function I Å k[1 0 exp(0TR/T1)] . A T2 mapwas obtained from a fit of 16 images obtained with a multiechosequence with TEs ranging from 0.014 to 0.224 s, with TR fixed

Figure 2. Photographs showing (A) neocartilage plug and (B) neo- at 10 s, to the functional form I Å k* exp(0TE/T2 ) .cartilage ribbon in the extracapillary space of a hollow fiber bioreactor The magnetization transfer rate between broad and narrow30 days postinoculation with cells. water resonances, corresponding roughly to bound and free water,

was calculated from the following equation: km Å 1/T1sat[1 0 Is /Io ] , where 1/T1sat denotes T1 in the presence of saturation [7,17] .

Figure 3. Histologic sections of cartilage plug stained with Alcian blue. Sections were cut perpendicular to the fiber axis and areas ofmetachromasia indicate high proteoglycan concentrations. Overall diameter of the sample is 4 mm. Original objective magnifications are: (A)15; (B) 110; (C) 120; (D) 140.

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The values Is and Io denote, respectively, the amplitude of the was very characteristic of hyaline cartilage. In certain areas, itappeared that the matrix had undergone extensive degradation,narrow resonance in the presence or absence of a 5-s, 12-mT

saturating pulse applied 6 kHz away from the narrow resonance. resulting in fusion between individual lacunae and a considerablereduction in the amount of extracellular matrix. The most promi-T1sat was obtained by fitting a series of images, acquired with

saturating pulses of variable duration, tpÅ 0.01–5 s, to the appro- nent site of matrix degradation was found at the periphery of thetissue sample [Fig. 3(C)]. The outer surface of the tissue whichpriate functional form [7] .

To obtain the T1r map of the HFBR, transverse magnetization had been in contact with the inner surface of the bioreactor con-sisted of a stromal layer of one to three flattened cells. Eachwas produced with a nonselective pulse and then spin locked

along the y-axis with an 8-mT pulse for a variable spin-lock time growth unit was also bounded by a similar stromal layer. Theboundary between the growth units surrounding the central and(SL). The map was calculated from a series of spin-echo images

acquired after SL ranging between 0.025 and 0.5 s [14,15]. lowermost fibers in Figure 3(B) consisted of a fusion of thesestromal layers. Certain regions of each growth unit containedabundant metachromatic matrix due to the presence of aggrecanF. 31P Spectroscopy. 31P spectra were acquired with a home-proteoglycan containing chondroitin sulfate side chains. The sur-built double resonance surface coil (diam. 10 mm) tunable toface of the tissue in contact with each fiber consisted of a thinboth the 31P and 1H frequencies. Spectra were obtained from thelayer of flattened cells similar to what was observed on the outerFourier transform of 34,000 signal-averaged free induction de-perimeter of each growth unit. In addition, basophilic staining wascays (FIDs) detected following a pseudo 907 pulse; a sweepnoted within the wall of each fiber suggesting that a certain amountwidth of 13 kHz was used and 4K data points were collected.of proteoglycan was entering the fiber. From the staining pattern,The interpulse delay was 2 s, and matched filtering with 40 Hz ofwe see that proteoglycan deposition was consistently low at theline broadening was used. A capillary tube containing methylene-stromal layers, including a layer just outside of the hollow fibers,diphosphonic acid (MDP) was attached to the outside of thewhile the greatest deposition was an irregularly shaped region withbioreactor and used as an external chemical shift reference, andlow cellularity located at the upper left in Figure 3(A).chemical shifts are reported relative to phosphocreatine (PCr).

In this preliminary work, we obtained only a single time pointRelative peak heights were assumed proportional to metabolitein the development of the tissue. However, based on chondrocyteconcentrations and intracellular pH determinations were basedgrowth properties and the geometry we observed, we conjectureon the pH-dependent chemical shift of inorganic phosphate (Pi)that the cells initially adhere to the fibers, forming a stromal layer[10–12].at the surface. These initial cells then proliferate radially withmore mature cells farthest from the fibers. When the growth units

III. RESULTS AND DISCUSSIONcontact each other, the stromal cells on the outer edges merge to

A. Morphology and Development of Neocartilage in the produce a thin interfacial junction of fibrous tissue. The fusionHFBR. Two distinct forms of neocartilage were produced. In the of two growth units noted above may be the result of cellularplug form [Fig. 2(A)], the cells filled in the extracapillary space proliferation between the two fibers.uniformly. In the ribbon form [Fig. 2(B)], the neocartilage filledthe bioreactor in a nonuniform fashion. The factors that lead to C. Biochemical Assays. Measurements of perfusate glucosethe production of one or the other of these morphologies are not and lactate concentrations were performed up to 17 days postinoc-entirely clear, but the ribbon formation may be related to a longer ulation. Average daily glucose utilization and lactate productiondelay between inoculation of cells and initiating perfusion of the during the intervals between measurements are presented in Fig-bioreactor. ure 4. The lactate-to-glucose ratio was small at the initial time

By visual inspection approximately 10 days after inoculation, point, but rapidly increased after 2 days, and subsequently re-the tissue in the reactor appeared opaque, and the media within mained essentially constant. These results are consistent with anthe extracapillary space appeared yellow, indicating a buildup oflactic acid. Two weeks after inoculation, the fibers were seen tobe completely encircled by neocartilage.

All imaging and spectroscopy studies presented here wereperformed on the pluglike cartilage, with the imaging plane ap-proximately 2 cm from the left edge of the reactor. Because thefiber arrangement along the length of the reactor is not entirelyuniform, some partial volume effects are expected in the NMRimages.

B. Histology. Histologic sections obtained after proton microi-maging at 30 days are shown in Figure 3. The hollow fibers arereadily identifiable in cross section. Each fiber was surroundedby a discrete region which we termed a growth unit, as seen inFigure 3(A,B) . The degree of cellularity in general was high, andthe cells had the appearance of chondrocytes located in discretelacunae. The cells in their lacunae were seen to be largely ar- Figure 4. Bar graph of glucose utilization and lactate production ofranged in a columnar pattern perpendicular to the fiber, although the tissue in the HFBR. Measurements were made in four bioreactorsthere was wide variability in the pattern [Fig. 3(B,D)] . The over the period of time up to 17 days postinoculation. Errors shown

are standard deviations.combination of the basophilic matrix and the cellular phenotype

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Figure 5. Proton NMR maps of (A) r, (B)T1, (C) T2 , (D) km , and (E) T1r . All imageswere obtained 30 days postinoculation withthe imaging plane oriented perpendicular tothe fiber axis. The in-plane resolution was 60mm, the slice thickness was 2 mm, and theFOV was 1.5 cm.

early transition from largely aerobic metabolism to a more mixed D. Proton NMR Microimaging. Proton NMR microimagingwas performed 30 days postinoculation. All measured parameterspicture in which glycolysis contributes significantly, as observed

by other authors using different techniques [18]. It is notable represent a spatial average over the imaging volume of 60mm 1 60 mm 1 2 mm. Owing to the high cellularity of thethat in the present experiments, the transition occurs well before

a significant amount of matrix has accumulated, suggesting that neocartilage in the HFBR, as compared to mature cartilage, im-aging parameters reflect combined properties of the matrix andthe increase in glycolysis may not be a result of oxygen starvation

of chondrocytes, but rather may be developmental in nature. The chondrocytes. Both calculated maps (Fig. 5) and weighted im-ages (Fig. 6) are shown. We did not obtain images immediatelymaximum in both glucose utilization and lactate consumption are

seen in the assay performed at day 11, suggesting that this may after inoculation, to avoid interfering with the adherence of thechondrocytes to the hollow fibers.be the time of maximum cell density or maximum metabolic

activity. It is also at approximately this time that the bioreactor The density images, Figures 5(A) and 6(A), show the overalldistribution of mobile protons. The fiber walls are clearly seenbecomes opaque owing to matrix accumulation.

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Figure 6. Weighted proton NMR images.The main contrast parameter and relevanttimings for each image are (A) r: TR Å 10 s,TE Å 13.5 ms; (B) T1 : TR Å 1.5 s, TE Å 13.0ms; (C) T2 : TRÅ 10 s, TEÅ 56 ms; (D) MTC:TR Å 10 s, TE Å 13.5 ms, saturation pulsetime Å 5 s; and (E) T1r : TR Å 10 s, TE Å13.5 ms, SL Å 100 ms. All images were ob-tained 30 days postinoculation with the im-aging plane oriented perpendicular to the fi-ber axis. In-plane resolution, slice thickness,and FOV are as in Figure 5.

as dark zones, as are the peripheries of the growth zones which The T1 map in Figure 5(B) shows that the less-hydrated tissuebetween growth units has a T1 value which is lower than thosecorrespond to the stromal layers seen in the histologic prepara-

tions. The remainder of the tissue shows no significant density of the surrounding neocartilage. Quantification of stromal layerT1s is difficult owing to the thinness of the layer. Typical T1variations. By comparison with the signal intensity within the

perfusate-filled hollow fibers, we estimate that the water content values for neocartilage were 2.5 s, as compared with a perfusateT1 of 3.5 { 0.1 s. This rather large value probably reflects theof the neocartilage surrounding the fibers was typically 80 { 5%.

Figure 5(A) provides a convenient reference point to assess presence of proteoglycan monomers in the perfusate, as con-firmed by gel electrophoresis (data not shown). A highly T1-the effect of rf inhomogeneity on displayed image intensities. By

comparing the intraluminal regions of the hollow fibers, we have weighted image would ideally show an intensity pattern oppositeto that of the T1 map; this is not seen in Figure 6(B), owing toconfirmed that the variation in image intensity due to rf inhomo-

geneity is approximately 15%. The tissue signal itself varies ap- the unavoidable weighting by r and T2 , both of which, in thiscase, operate to counter the contrast imposed by the T1 weighting.proximately 20% in Figure 5(A). Thus, the tissue signal is quite

homogeneous, with a variation on the order of 5%, in terms of The T2 map is consistent with the T1 map, showing larger T2sin regions with high water content. In addition, the T2 imagesaverage spin density.

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Figure 7. 31P-NMR spectra of a bioreactor inoculated with 107 cells acquired (A) 10 days and (B) 17 days postinoculation. The labeledresonances are: PME Å phosphomonoesters; Pi Å inorganic phosphate; PCr Å phosphocreatine; a, b, and g ATP Å the three phosphorusnuclei of ATP; DPDE Å diphosphodiesters.

show a significant amount of heterogeneity within the tissue. from proteoglycans. However, histochemical studies in both ma-ture and developing cartilage have demonstrated that collagenComparing the T2 images with the density images, it can be seen

that while the perfusate is bright in all four of these images, the and proteoglycans are typically not spatially separated. It is possi-ble that the results published elsewhere [6,9], based on selectivefiber walls, while dark in the density images, are bright in the T2

images. This reflects mobile protons in the perfusate traversing enzymatic digestion of mature cartilage, do not apply to ournative neocartilage.the fiber walls. Just outside the fibers, where the density map

becomes bright, the T2 map is dark. We interpret this as reflecting The predominant origin of T1r contrast is from molecular mo-tions at frequencies comparable to the spin-lock frequency. Thus,high proton density but short T2s of the light-staining regions just

outside the hollow fibers [Fig. 3(B,D)]. Typical T2 values are, both the T1r map, Figure 5(E), and the T1r-weighted image,Figure 6(E), appear bright for regions with fast motion, and darkfor the perfusate, 58 { 2 ms; for the stromal cells, 22.9 { 3.5

ms; and for the large proteoglycan deposit noted in the histology for regions with components at very low frequencies [14,15] .We can see from these figures that the stromal layer betweensection above, 43{ 4 ms. This latter value is larger than expected,

given the low cellularity of the region. We interpret this as indi- growth units and the light-staining layer just outside the hollowfibers have a large component of water which is relatively fixedcating the presence of matrix with a higher concentration of free

water than is seen in mature cartilage. This may reflect the dy- in comparison with the more cellular regions of the neocartilage.namic nature of the neocartilage system.

Considerable tissue heterogeneity was also seen in the magne- E. 31P Spectroscopy. Representative 31P spectra of the HFBRtization transfer rate images [Figs. 5(D) and 6(D)]. The local obtained at days 10 and 17 postinoculation are shown in Figurekm values are shown in Figure 5(D), with larger km being brighter 7. The spectra are essentially identical, demonstrating metabolicand smaller km being darker. The km reflects the rate of the transfer stability of the preparation over this time period. The resonancesof magnetization from the relatively fixed protons in collagen to correspond to the a, b, and g phosphate groups of adenosinethe more mobile protons in interstices.

The darkest regions (lowest km values) in the extracapillaryspace corresponded to areas of high proteoglycan concentrations,

Table I. 31P NMR measured metabolic constants for neocartilage.as seen in the middle regions of the growth units and in the regionof dense proteoglycan deposition to which we have previously Metabolic Data Day 10 Day 17referred. In contrast, the stromal layers and the light staining

ATP/Pi 2.58 2.24region around the hollow fibers are seen to be relatively brightPCr/Pi 1.04 0.91in the km map. Previous work [6,9] has suggested that MTC inpH-intracellular 7.23 6.98cartilage is dominated by collagen, with only a minor contribution

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triphosphate (ATP), PCr, Pi, phosphomonoesters (PME), and for their technical advice throughout. The authors also acknowledgeElsie Williams for her help in preparing the figures.diphosphodiesters (DPDE) [10–12]. Chemical shifts are labeled

relative to that of PCr, and resonance heights depict relativeREFERENCESmetabolite concentrations. As the a and g ATP resonances con-1. M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L.tain small contributions from the a and b phosphorus nuclei of

Petersen. ‘‘Treatment of deep cartilage defects in the knee with autologousadenosine diphosphate, we used the b-ATP resonance to deter-chondrocyte transplantation,’’ N. Engl. J. Med. 331, 889–895 (1994).mine the ATP/Pi ratios for neocartilage tissue at 10 and 17 days

2. P. T. Callaghan, Principles of Nuclear Magnetic Resonance Micros-postinoculation (Table I) . These spectra were obtained with TRcopy (Oxford Science, Oxford, UK), 1991.

of 2 s to improve signal-to-noise (S/N), so that the resonances 3. L. E. Freed, G. Vunjak-Novakovic, and R. Langer. ‘‘Cultivation of cell-are saturated to different degrees. In the absence of T1 measure- polymer implants in bioreactors,’’ J. Cell. Biochem. 51, 257–264 (1993).ments for similar preparations, we approximate T1 (PCr) É 3 s, 4. L. C. Gerstenfeld and W. J. Landis. ‘‘Gene expression and extracellu-T1 (b-ATP) É 0.75 s, and T1 (Pi) É 2 s, using the fact that in lar matrix ultrastructure of a mineralizing chondrocyte cell culturenumerous studies, T1 (b-ATP) É T1 (g-ATP) [16]. Therefore, system,’’ J. Cell Biol. 12, 501–513 (1991).

5. R. J. Gillies, J.-P. Galons, K. A. McGovern, P. G. Scherer, Y.-H. Lien,the actual concentrations of PCr and Pi with respect to g-ATP areC. Job, R. Ratcliff, F. Chapa, S. Cerdan, and B. E. Dale. ‘‘Designapproximately twice and 1.5 times that evident from the spectrum,and application of NMR-compatible bioreactor circuits for extendedrespectively.perfusion of high-density mammalian cell cultures,’’ NMR Biomed.We note that between days 10 and 17 postinoculation, the6, 95–104 (1993).chemical shift difference of Pi peak relative to PCr decreased

6. M. L. Gray, D. Burstein, L. M. Lesperance, and L. Gehrke. ‘‘Magneti-from 5.07 to 4.72 ppm. The corresponding decrease in intracellu- zation transfer in cartilage and its constituent macromolecules,’’lar pH, from 7.23 to 6.98, is consistent with the known increase Magn. Reson. Med. 34, 319–325 (1995).in glycolytic metabolism that occurs in developing chondrocyte- 7. J. V. Hajnal, C. J. Baudouin, A. Oatridge, I. R. Young, and G. M. By-cartilage systems. dder. ‘‘Design and implementation of magnetization transfer pulse se-

It is important to emphasize that our goal was to optimize quences for clinical use,’’ J. Comput. Assist. Tomogr. 16, 7–18 (1992).8. W. Horton and J. R. Hassell. ‘‘Independence of cell shape and loss ofconditions for cell growth and matrix expression. This required

cartilage matrix production during retinoic acid treatment of cultureda relatively low number of cells, 107 , to be inoculated. Neverthe-chondrocytes,’’ Dev. Biol. 115, 392–397 (1986) .less, we obtained 31P spectra which had comparable S/N to spec-

9. D. K. Kim, T. L. Ceckler, V. C. Hascall, A. Calabro, and R. S. Bala-tra presented in other reports in which a significantly greaterban. ‘‘Analysis of water-macromolecule proton magnetization trans-number of cells was used [10,12].fer in articular cartilage,’’ Magn. Reson. Med. 29, 211–215 (1993) .

10. B. J. Kvam, P. Pollesello, F. Vittur, and S. Paoletti. ‘‘ 31P NMR studiesof resting zone cartilage from growth plate,’’ Magn. Reson. Med. 25,IV. CONCLUSIONS355–361 (1992) .

We have succeeded in growing cartilage tissue in the extracapil- 11. R. B. Moon and J. H. Richards. ‘‘Determination of intracellular pHlary space of an NMR-compatible hollow fiber bioreactor. This by 31P magnetic resonance,’’ J. Biol. Chem. 248, 7276–7278 (1973) .system is well-suited to serial noninvasive spectroscopic and im- 12. P. Pollesello, B. Bernard, M. Grandolfo, S. Paoletti, F. Vittur, and

B. J. Kvam. ‘‘Energy state of chondrocytes assessed by 31P-NMRaging studies of chondrocyte metabolism, development and ma-studies of preosseous cartilage,’’ Biochem. Biophys. Res. Commun.trix production. We are presently in the process of investigating180, 216–220 (1991).the metabolic profile of chondrocytes at different stages of growth

13. G. Quintarelli, J. E. Scott, and M. C. Dellovo. ‘‘The chemical andas well as flux rates through enzymes central to intracellularhistochemical properties of Alcian blue II. Dye binding of tissuebioenergetics. We are also further defining the correlation ofpolyanions,’’ Histochemie 4, 86–98 (1964).

NMR properties with macromolecular composition. 14. G. E. Santyr, E. J. Fairbanks, F. Kelcz, and J. A. Sorenson. ‘‘Off-The importance of further investigation of neocartilage is high- resonance spin locking for MR imaging,’’ Magn. Res. Med. 32, 43–

lighted by the potential of chondrocyte transplantation as a means 51 (1994).for treating degenerative joint disease [1,3] . The HFBR system 15. R. E. Sepponen, T. Pohjonen, J. T. Sipponen, and J. I. Tanttu. ‘‘Amay be a powerful tool for detailed exploration of cartilage regen- method for T1r imaging,’’ J. Comput. Assist. Tomogr. 9, 1007–1011

(1985) .eration in vivo, including the effects of growth factors to promote16. R. G. S. Spencer, J. A. Balschi, J. S. Leigh, and J. S. Ingwall. ‘‘ATPdevelopment and to counteract specific naturally produced growth

synthesis and degradation rates in the perfused rat heart,’’ Biophys.inhibitors.J. 54, 921–929 (1988). R. G. S. Spencer, J. A. Balschi, J. S. Leigh,and J. S. Ingwall. ‘‘Erratum,’’ Biophys. J. 55, 209 (1989) .

17. J. Eng, T. L. Ceckler, and R. S. Balaban. ‘‘Quantitative 1H magnetiza-ACKNOWLEDGMENTStion transfer imaging in vivo,’’ Magn. Reson. Med. 17, 304–314

The authors thank Cellco, Inc., for the use of their Cell Max Quad (1991) .pump; Malcom Morrison at Microgon, Inc., for supplying the fibers 18. H. Zipper, S. K. Papierman, R. M. Libbin, and P. Person. ‘‘Develop-used in our bioreactors; and Ciba-Geigy Corp. for their donations ment of chick limb bud chondrocytes in cell culture: Morphologicof essential laboratory equipment. They are grateful to Louis Gers- and oxidative metabolic observations,’’ Clin. Orthop. Rel. Res. 155,

186–195 (1981) .tenfeld, William Landis, Elizabeth O’Byrne, and Ronald Goldberg

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