TISSUE ENGINEERING

Volume 9 Number 1 2003

copy Mary Ann Liebert Inc

Functional Assessment of Tissue-Engineered Meniscal

Cartilage by Magnetic Resonance Imaging and Spectroscopy

ANDREacute A NEVES ME1 NICK MEDCALF ME2 and KEVIN BRINDLE PhD1

ABSTRACT

A perfusion bioreactor system was used to grow bioartificial meniscal cartilage tissue in vitro Mag-netic resonance imaging and magnetic resonance spectroscopy methods were used to characterizethe flow and perfusion profiles and the growth distribution and bioenergetics of the fibrochon-drocytes in the resulting constructs These measurements were correlated with each other and withsubsequent histologic analysis The study has demonstrated that these noninvasive magnetic reso-nance methods will be useful for designing bioreactor operation strategies and cell scaffolds thatlead to the production of tissue-engineered meniscal cartilage constructs with properties resemblingthose of the native tissue

51

INTRODUCTION

CARTILAGE DEGENERATION due to primary osteoarthri-

tis or trauma is a major cause of disability in mid-

dle-aged and older people1 The relative inability of the

tissue to self-repair means that these injuries are main-

tained for years and can eventually lead to further de-

generation (secondary osteoarthritis) Treatment depends

on the nature and extent of the injury For example some

tears in meniscal cartilage can be repaired simply by su-

turing and relatively small areas of damaged articular car-

tilage have been repaired by cell transplantation2 How-

ever where the loss of tissue is more extensive treatment

frequently requires tissue transplantation Tissue grafts

can be autografts in which cartilage is replaced with

small tissue plugs from the same or another joint in the

same individual or full allografts in which tissue is trans-

planted from another individual The problem with the

former approach is that transplantable material is limited

and concerns over disease transmission and immuno-

genicity have limited the latter3 These limitations have

stimulated the development of tissue-engineered struc-

tures that mimic the function of native tissue and that

could be used in the repair of larger cartilage defects

These have included three-dimensional scaffolds based

on synthetic4 or natural polymers5 which have been im-

planted either alone or after seeding with chondrocytes

There has been significant progress in growing carti-

lage tissues in vitro with properties similar to those found

in vivo However the scale-up of this technology to a

clinical setting remains a significant problem6 Several

bioreactor systems have been used for cartilage produc-

tion including spinner flasks7 and perfusion cultures8 In

the case of the latter the constant availability of fresh

medium the mechanical action of shear stress and the

ability to transport nutrients through an increasingly

dense mass of cells and extracellular matrix material has

favored their use9 However the optimal flow parame-

ters and scaffold geometries for the generation of menis-

cal tissue in these systems have yet to be identified To

do this there is a need for methods that can be used to

assess in an intact and functioning reactor the effects of

perfusion and nutrient diffusion on reactor performance

in terms of cell growth and distribution and matrix pro-

duction

A suite of magnetic resonance imaging (MRI) and

1Department of Biochemistry University of Cambridge Cambridge United Kingdom2Smith amp Nephew Group Research Centre Heslington York United Kingdom

magnetic resonance spectroscopy (MRS) methods has

been developed for assessing the performance of in-

tensive mammalian cell bioreactor systems These in-

clude methods for measuring cell growth and distribu-

tion10 cell volume1112 the distribution of oxygen13

and cellular metabolism14 and methods for measuring

nutrient flow and diffusion15 We show here that these

methods can be used to monitor and optimize the per-

formance of a perfusion bioreactor system for growing

meniscal cartilage in vitro In comparison with articu-

lar cartilage relatively little work has been done on

the in vitro synthesis of tissue-engineered meniscal

constructs6

MATERIALS AND METHODS

Cell subculture routine and medium composition

Sheep meniscal fibrochondrocytes were supplied by

Smith amp Nephew Group Research Centre (SampN GRC

Heslington York UK) as primary cells (P0) These

were subsequently propagated in static culture flasks to

the fourth passage (P4) to ensure consistency of the

bioreactor inoculum Cells were grown in Dulbeccorsquos

modified Eaglersquos medium containing glucose (45 g

L21) L-glutamine (584 mg L21) 10 fetal bovine

serum 10 mM HEPES 01 mM nonessential amino

acids and gentamicin (20 mg L21) (G medium) a mod-

ification of a medium composition proposed else-

where16 The cells were seeded at a density of 3 3 104

cells cm22 in fresh medium for propagation and split

when they reached 70 confluency Production

medium (P) which stimulates the production of extra-

cellular matrix material consisted of the same compo-

nents as in G medium but also included 04 mM L-pro-

line and ascorbic acid (50 mg mL21) in the form of

ascorbate phosphate16

Scaffold properties and seeding method

Scaffolds consisting of 15-mm-diameter polyethylene

terephthalate (PET) fibers with a void volume of 97 and

a density of 45 mg cm23 were supplied in the form of

disks 12 mm in diameter and 4 mm thick by SampN GRC

The scaffolds were seeded with P4 sheep fibrochondro-

cytes in well-mixed 250-mL spinner flasks (Fisher Scien-

tific Pittsburgh PA) using a method described else-

where17 Each flask was inoculated with 96 3 107

fibrochondrocytes corresponding to 12 3 107 cells per

scaffold Over a period of 3 days cells attached to the sur-

face of the scaffolds with no significant cell loss and an

adhesion yield greater than 95 The scaffolds were then

transferred aseptically to the bioreactor and perfused at a

number of different flow rates for periods of up to 2 weeks

NEVES ET AL

Bioreactor setup and operation

The system used here is a modification of that used

previously11 Each of the bioreactors which were de-

signed and custom-made in-house consisted of a poly-

sulfone (RS Components Northants UK) tube (20-mm

id) with a capped cylindrical chamber at the top (40-

mm id) The fixed bed consisted of three seeded scaf-

folds positioned perpendicularly to the ascending flow of

medium The lower section of the bioreactor was fitted

with plastic spacers (ultra-high-density polyethylene RS

Components) which allowed for separation of the scaf-

folds and for flow of medium both through and around

the scaffolds (Fig 1) The presence of the flow-diverting

slots at the edges of the spacers prevented excessive

build-up of pressure as the scaffolds became filled with

cells and matrix material The complete system was as-

sembled under aseptic conditions in a laminar flow hood

The bioreactors were then perfused with fresh production

medium (P) at the specified flow rates Medium was re-

placed continuously in a conditioning vessel at a dilution

rate of 025 day21 Ascorbate phosphate was added every

2ndash3 days16 at a concentration of 50 mg mL21

Magnetic resonance imaging and magneticresonance spectroscopy methods

Magnetic resonance imaging and spectroscopy were

performed with a vertical wide-bore Oxford Instruments

(Oxford UK) magnet (94 T 89-cm bore diameter)

equipped with an unshielded gradient set interfaced to a

Varian (Palo Alto CA) UnityPlus spectrometer con-

trolled by a SUN SPARCstation IPX running VNMR

53B software 1H spectra and images were acquired at

400 MHz with a Varian 25-mm 1H imaging probe and31P spectra were acquired at 1613 MHz with a Bruker

25-mm 1H31P probe

Diffusion-weighted MRI

Diffusion-weighted MR images were acquired with a

stimulated echo (STEAM) sequence as described previ-

ously11 An echo time (TE) of 40 ms and pulsed magnetic

field gradients of 02 T m21 and 25-ms duration were

used The mixing time (TM) was 03 s providing a dif-

fusion weighting (b) of 564 3 109 rad2 s m22 The field-

of-view was 25 3 25 mm acquired into 64 3 128 data

points giving an in-plane resolution of 01 3 04 mm

Measurement of construct perfusion using acontrast agent

A 10 mM solution of the contrast agent gadolin-

ium(III)-diethyltriaminepentaacetic acid (Gd-DTPA)

(Magnevist Schering West Sussex UK) was added to

the perfusion medium in the conditioning vessel and

52

contrast agent inflow into the constructs was observed

with a series of T1-weighted spin-echo images The ac-

quisition parameters for these images were TR 5 130 ms

TE 5 123 ms and the slice thickness was 20 mm The

field-of-view was 20 3 20 mm acquired into 512 3 128

(phase encode) data points giving an in-plane resolution

of 008 3 016 mm Maps of the paramagnetic contribu-

tion to the relaxation rate (R1p) were derived for each of

the T1-weighted images in the time course1115 R1p is di-

rectly proportional to the concentration of the contrast

agent

MRI measurements of flow

Axial flow through and around the scaffolds was mea-

sured by a time-of-flight MR imaging method This was

based on a selective inversion recovery pulse sequence in

which slice-selective spin-tagging and detection pulses

were followed by a bipolar readout gradient18 Flow rates

were determined on a pixel-by-pixel basis from the flow-

dependent changes in the apparent T1 The acquisition

parameters for these images were TR 5 01 ms TE 5

273 ms and the slice thickness was 30 mm Images were

acquired at 12 different delays ranging from 000625 to

64 s between the 180deg slice-selective inversion pulse and

the low flip-angle slice-selective detection pulse This

range of delays was chosen to ensure full recovery of the

water proton magnetization to its equilibrium state both

in the presence and absence of flow The field-of-view

was 25 3 25 mm acquired into 64 3 64 (phase encode)

data points giving an in-plane resolution of 02 3

MRI ANALYSIS OF MENISCAL CARTILAGE

04 mm Four transients were acquired per phase encode

increment giving a total image acquisition time of 40 s

31P magnetic resonance spectroscopymeasurements of cellular energy metabolism

31P nuclear magnetic resonance (NMR) spectra of the

bioreactors were acquired as described previously11 A

40-ms 90deg pulse and a repetition time of 14 s were used

Histologic analysis

Constructs were harvested from the bioreactors at the

end of the cultivation period of 2 weeks and immersed

in a 4 solution of formaldehyde before histologic ex-

amination Samples were later dehydrated with graded

concentrations of ethanol and embedded in glycol

methacrylate (GMA) resin using an embedding kit

(Technovit 7100 TAA Laboratories Equipment Alder-

maston UK) Blocks were sectioned and histologic sec-

tions (8 mm thick) were produced by an automated mi-

crotome with disposable tungsten carbide knives

Sections were stained with Mayerrsquos hematoxylin and

phloxine B (Sigma Dorset UK)

Collagen and glycosaminoglycan analysis

The glycosaminoglycan (GAG) content of the samples

was determined spectrophotometrically using the di-

methylmethylene blue dye (DMB) method19 Total col-

lagen content was determined from the measured hy-

droxyproline content of the constructs after acid

hydrolysis and reaction with p-dimethylaminobenzalde-

hyde and chloramine-T20 using a ratio of hydroxypro-

line to collagen of 014321

RESULTS

Cell distribution and content

The time-dependent changes in the signal intensities

in diffusion-weighted images of scaffolds perfused at two

different flow rates are shown in Fig 2 These changes

parallel to some extent changes in the nucleoside

triphosphate (NTP) content of the scaffolds determined

by 31P MRS (Fig 2B) as has been observed previously

for CHO cells growing in a fixed-bed bioreactor11 and is

consistent with signal intensity in the diffusion-weighted

MR image being primarily a measure of cell content

Both sets of data indicate that at 30 mL min21 there was

a progressive increase in cell content over the 2 weeks

of culture However at 60 mL min21 there was an ini-

tial increase in cell content followed by a subsequent de-

cline in the second half of the culture which was ob-

served in the diffusion-weighted images as a loss of signal

intensity at the center of the scaffold (Fig 2A)

53

The T1 or spin-lattice relaxation time is the time constant

for recovery of the bulk magnetization in an NMR experiment

to its equilibrium value following a perturbation The relaxation

rate (R1) is the inverse of this (R1 5 1T1) In the T1-weighted

imaging experiment used here signal intensity is directly pro-

portional to the relaxation rate This in turn is directly pro-

portional to the concentration of the contrast agent Gd-DTPA

which enhances spin-lattice relaxation

FIG 1 Bioreactor design (A) Flow cell with cell scaffold

(indicated by cross-hatching) located between two supporting

disks (B) Top view of a supporting disk The slots around the

edge allow for diversion of flow away from the construct as

this becomes progressively blocked with cells and extracellu-

lar matrix material

Scaffold perfusion

Perfusion of the scaffolds was measured by adding

a contrast agent (Gd-DTPA molecular weight 590

gmol21) to the medium This paramagnetic agent which

is used in clinical imaging to enhance tissue contrast and

to measure perfusion produces an increase in signal in-

tensity in T1-weighted images Analysis of these changes

in intensity can be used to produce concentration maps

of the contrast agent15 Thus by acquiring a series of im-

ages following injection of the contrast agent the per-

fusion of the scaffolds can be monitored This is illus-

trated in Fig 3 which shows a series of T1-weighted

images acquired from a scaffold after injection of the

contrast agent on days 7 and 14 after cell seeding The

increase in signal intensity approximately 6 min after

contrast agent injection indicated arrival of the agent in

the spaces around the scaffold There was then an in-

crease in signal within the scaffold as it became infil-

trated by the contrast agent The rate of contrast agent

NEVES ET AL

inflow into the scaffold was clearly faster on day 7 than

on day 14 The arrow in Fig 3A indicates a region that

enhanced relatively rapidly This contained a needle hole

that was made in the scaffold during cell seeding The

needle hole had become blocked by day 14 Figure 4

shows the time-dependent changes in contrast agent con-

centration in five different regions of interest (ROIs) in

the two different scaffolds perfused at different flow rates

(30 and 60 mL min21) Four of these ROIs were within

the volume of the construct (ROI1 to ROI4 labeled in

the direction of medium flow) and the fifth was imme-

diately above it These perfusion profiles were obtained

immediately after cell seeding and on days 7 and 14 of

cultivation The perfusion profiles obtained immediately

after cell seeding of the four internal ROIs and the ex-

ternal ROI were similar to each other and appeared to be

independent of the flow rate The similarity of the pro-

files for the external and internal ROIs coupled with the

direct measurements of linear flow velocities within the

construct (see below) showed that penetration of the con-

54

FIG 2 (A) Diffusion-weighted MR images acquired from a cell-seeded scaffold during a bioreactor run The image plane was

within the construct and was parallel to the direction of flow Signal intensity is inversely proportional to the ADC of water that

is high signal intensity corresponds to a higher cell content (see text) (i) Flow rate 5 30 mL min21 (ii) flow rate 5 60 mL

min21 (B) (i) Relative time-dependent changes in the average signal intensity in the diffusion-weighted images shown in (A)

and (ii) the NTP content of the constructs determined by 31P MRS (s) Flow rate 5 30 mL min21 (j) flow rate 5 60 mL min21

trast agent into the scaffold at this stage of the culture

was driven primarily by flow rather than by diffusion

However by day 7 in the scaffold perfused at a flow rate

of 30 mL min21 the rate of inflow of contrast agent was

much reduced and this was further reduced by day 14

Closer examination of the perfusion profiles obtained on

days 7 and 14 shows that the regions exposed directly

(ROI1) or indirectly (ROI4) to the flow were now more

efficiently perfused than the internal ROIs This indicates

that diffusion rather than flow is now primarily respon-

sible for inflow of the contrast agent into the construct

The diffusion coefficient for the contrast agent in the

outer ROIs was determined by using a simple one-di-

mensional non-steady-state diffusion model (infinite slab

model) proposed by Crank22 (see Fig 5A) We have used

this model previously to determine the diffusion coeffi-

cient of Gd-DTPA in cell-filled carriers in a fixed-bed

bioreactor15 A diffusion coefficient of (7 6 05) 3

10211 m2 s21 was determined for ROI1 which is simi-

lar to a value of 92 3 10211 m2 s21 found by Foy and

Blake23 for the diffusion of Gd-DTPA in native human

articular cartilage

The value of R1p which is directly proportional to the

concentration of the contrast agent can be used to esti-

mate the volume fraction of the construct from which the

agent is excluded15 By day 14 at a flow rate of 30 mL

MRI ANALYSIS OF MENISCAL CARTILAGE

min21 the excluded volume fraction was estimated at

55 (Fig 5B) Removal of the construct at this stage and

subsequent cell counting gave a cell volume fraction of

53 assuming that a fibrochondrocyte has a volume of

1028 cm3 (estimated by using a calibration of cell num-

ber vs wet weight results not shown) The collagen and

GAG contents were measured at 57 and 11 of the fi-

nal dry weight respectively (or 06 and 011 of the

final wet weight) and thus could make only a negligible

contribution to the excluded volume fraction Measure-

ments of NTP content and cell counting indicated that at

this flow rate the cell number in the scaffold increased

by nearly a factor of 2 during the 14 days of the culture

Yet the excluded volume data suggested that there was a

nearly 7-fold increase in cell content from 8 on day 1

to 55 on day 14 This underestimate of the excluded or

cell volume fraction on day 1 is due to the effects of flow

which by decreasing the apparent T1 increased the esti-

mated R1p or Gd-DTPA concentration in the scaffold

However by day 14 the increase in cell number and de-

position of matrix material has reduced flow through the

construct to undetectable levels (see below) and thus the

measured water T1 in the construct is now a true reflec-

tion of the Gd-DTPA concentration This also explains

why at a flow rate of 60 mL min21 at which there is al-

ways macroscopic flow of material through the construct

55

FIG 3 Time course of Gd-DTPA inflow in a construct on day 7 (A) and day 14 (B) of the bioreactor run The imaging plane

was parallel to the direction of flow and perpendicular to the plane of the construct The medium flow rate was 30 mL min21

The arrow indicates the location of the hole made in the scaffold during the seeding procedure The numbers indicate the time

(minutes seconds) after injection of the contrast agent into the medium conditioning vessel (see Materials and Methods)

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

magnetic resonance spectroscopy (MRS) methods has

been developed for assessing the performance of in-

tensive mammalian cell bioreactor systems These in-

clude methods for measuring cell growth and distribu-

tion10 cell volume1112 the distribution of oxygen13

and cellular metabolism14 and methods for measuring

nutrient flow and diffusion15 We show here that these

methods can be used to monitor and optimize the per-

formance of a perfusion bioreactor system for growing

meniscal cartilage in vitro In comparison with articu-

lar cartilage relatively little work has been done on

the in vitro synthesis of tissue-engineered meniscal

constructs6

MATERIALS AND METHODS

Cell subculture routine and medium composition

Sheep meniscal fibrochondrocytes were supplied by

Smith amp Nephew Group Research Centre (SampN GRC

Heslington York UK) as primary cells (P0) These

were subsequently propagated in static culture flasks to

the fourth passage (P4) to ensure consistency of the

bioreactor inoculum Cells were grown in Dulbeccorsquos

modified Eaglersquos medium containing glucose (45 g

L21) L-glutamine (584 mg L21) 10 fetal bovine

serum 10 mM HEPES 01 mM nonessential amino

acids and gentamicin (20 mg L21) (G medium) a mod-

ification of a medium composition proposed else-

where16 The cells were seeded at a density of 3 3 104

cells cm22 in fresh medium for propagation and split

when they reached 70 confluency Production

medium (P) which stimulates the production of extra-

cellular matrix material consisted of the same compo-

nents as in G medium but also included 04 mM L-pro-

line and ascorbic acid (50 mg mL21) in the form of

ascorbate phosphate16

Scaffold properties and seeding method

Scaffolds consisting of 15-mm-diameter polyethylene

terephthalate (PET) fibers with a void volume of 97 and

a density of 45 mg cm23 were supplied in the form of

disks 12 mm in diameter and 4 mm thick by SampN GRC

The scaffolds were seeded with P4 sheep fibrochondro-

cytes in well-mixed 250-mL spinner flasks (Fisher Scien-

tific Pittsburgh PA) using a method described else-

where17 Each flask was inoculated with 96 3 107

fibrochondrocytes corresponding to 12 3 107 cells per

scaffold Over a period of 3 days cells attached to the sur-

face of the scaffolds with no significant cell loss and an

adhesion yield greater than 95 The scaffolds were then

transferred aseptically to the bioreactor and perfused at a

number of different flow rates for periods of up to 2 weeks

NEVES ET AL

Bioreactor setup and operation

The system used here is a modification of that used

previously11 Each of the bioreactors which were de-

signed and custom-made in-house consisted of a poly-

sulfone (RS Components Northants UK) tube (20-mm

id) with a capped cylindrical chamber at the top (40-

mm id) The fixed bed consisted of three seeded scaf-

folds positioned perpendicularly to the ascending flow of

medium The lower section of the bioreactor was fitted

with plastic spacers (ultra-high-density polyethylene RS

Components) which allowed for separation of the scaf-

folds and for flow of medium both through and around

the scaffolds (Fig 1) The presence of the flow-diverting

slots at the edges of the spacers prevented excessive

build-up of pressure as the scaffolds became filled with

cells and matrix material The complete system was as-

sembled under aseptic conditions in a laminar flow hood

The bioreactors were then perfused with fresh production

medium (P) at the specified flow rates Medium was re-

placed continuously in a conditioning vessel at a dilution

rate of 025 day21 Ascorbate phosphate was added every

2ndash3 days16 at a concentration of 50 mg mL21

Magnetic resonance imaging and magneticresonance spectroscopy methods

Magnetic resonance imaging and spectroscopy were

performed with a vertical wide-bore Oxford Instruments

(Oxford UK) magnet (94 T 89-cm bore diameter)

equipped with an unshielded gradient set interfaced to a

Varian (Palo Alto CA) UnityPlus spectrometer con-

trolled by a SUN SPARCstation IPX running VNMR

53B software 1H spectra and images were acquired at

400 MHz with a Varian 25-mm 1H imaging probe and31P spectra were acquired at 1613 MHz with a Bruker

25-mm 1H31P probe

Diffusion-weighted MRI

Diffusion-weighted MR images were acquired with a

stimulated echo (STEAM) sequence as described previ-

ously11 An echo time (TE) of 40 ms and pulsed magnetic

field gradients of 02 T m21 and 25-ms duration were

used The mixing time (TM) was 03 s providing a dif-

fusion weighting (b) of 564 3 109 rad2 s m22 The field-

of-view was 25 3 25 mm acquired into 64 3 128 data

points giving an in-plane resolution of 01 3 04 mm

Measurement of construct perfusion using acontrast agent

A 10 mM solution of the contrast agent gadolin-

ium(III)-diethyltriaminepentaacetic acid (Gd-DTPA)

(Magnevist Schering West Sussex UK) was added to

the perfusion medium in the conditioning vessel and

52

contrast agent inflow into the constructs was observed

with a series of T1-weighted spin-echo images The ac-

quisition parameters for these images were TR 5 130 ms

TE 5 123 ms and the slice thickness was 20 mm The

field-of-view was 20 3 20 mm acquired into 512 3 128

(phase encode) data points giving an in-plane resolution

of 008 3 016 mm Maps of the paramagnetic contribu-

tion to the relaxation rate (R1p) were derived for each of

the T1-weighted images in the time course1115 R1p is di-

rectly proportional to the concentration of the contrast

agent

MRI measurements of flow

Axial flow through and around the scaffolds was mea-

sured by a time-of-flight MR imaging method This was

based on a selective inversion recovery pulse sequence in

which slice-selective spin-tagging and detection pulses

were followed by a bipolar readout gradient18 Flow rates

were determined on a pixel-by-pixel basis from the flow-

dependent changes in the apparent T1 The acquisition

parameters for these images were TR 5 01 ms TE 5

273 ms and the slice thickness was 30 mm Images were

acquired at 12 different delays ranging from 000625 to

64 s between the 180deg slice-selective inversion pulse and

the low flip-angle slice-selective detection pulse This

range of delays was chosen to ensure full recovery of the

water proton magnetization to its equilibrium state both

in the presence and absence of flow The field-of-view

was 25 3 25 mm acquired into 64 3 64 (phase encode)

data points giving an in-plane resolution of 02 3

MRI ANALYSIS OF MENISCAL CARTILAGE

04 mm Four transients were acquired per phase encode

increment giving a total image acquisition time of 40 s

31P magnetic resonance spectroscopymeasurements of cellular energy metabolism

31P nuclear magnetic resonance (NMR) spectra of the

bioreactors were acquired as described previously11 A

40-ms 90deg pulse and a repetition time of 14 s were used

Histologic analysis

Constructs were harvested from the bioreactors at the

end of the cultivation period of 2 weeks and immersed

in a 4 solution of formaldehyde before histologic ex-

amination Samples were later dehydrated with graded

concentrations of ethanol and embedded in glycol

methacrylate (GMA) resin using an embedding kit

(Technovit 7100 TAA Laboratories Equipment Alder-

maston UK) Blocks were sectioned and histologic sec-

tions (8 mm thick) were produced by an automated mi-

crotome with disposable tungsten carbide knives

Sections were stained with Mayerrsquos hematoxylin and

phloxine B (Sigma Dorset UK)

Collagen and glycosaminoglycan analysis

The glycosaminoglycan (GAG) content of the samples

was determined spectrophotometrically using the di-

methylmethylene blue dye (DMB) method19 Total col-

lagen content was determined from the measured hy-

droxyproline content of the constructs after acid

hydrolysis and reaction with p-dimethylaminobenzalde-

hyde and chloramine-T20 using a ratio of hydroxypro-

line to collagen of 014321

RESULTS

Cell distribution and content

The time-dependent changes in the signal intensities

in diffusion-weighted images of scaffolds perfused at two

different flow rates are shown in Fig 2 These changes

parallel to some extent changes in the nucleoside

triphosphate (NTP) content of the scaffolds determined

by 31P MRS (Fig 2B) as has been observed previously

for CHO cells growing in a fixed-bed bioreactor11 and is

consistent with signal intensity in the diffusion-weighted

MR image being primarily a measure of cell content

Both sets of data indicate that at 30 mL min21 there was

a progressive increase in cell content over the 2 weeks

of culture However at 60 mL min21 there was an ini-

tial increase in cell content followed by a subsequent de-

cline in the second half of the culture which was ob-

served in the diffusion-weighted images as a loss of signal

intensity at the center of the scaffold (Fig 2A)

53

The T1 or spin-lattice relaxation time is the time constant

for recovery of the bulk magnetization in an NMR experiment

to its equilibrium value following a perturbation The relaxation

rate (R1) is the inverse of this (R1 5 1T1) In the T1-weighted

imaging experiment used here signal intensity is directly pro-

portional to the relaxation rate This in turn is directly pro-

portional to the concentration of the contrast agent Gd-DTPA

which enhances spin-lattice relaxation

FIG 1 Bioreactor design (A) Flow cell with cell scaffold

(indicated by cross-hatching) located between two supporting

disks (B) Top view of a supporting disk The slots around the

edge allow for diversion of flow away from the construct as

this becomes progressively blocked with cells and extracellu-

lar matrix material

Scaffold perfusion

Perfusion of the scaffolds was measured by adding

a contrast agent (Gd-DTPA molecular weight 590

gmol21) to the medium This paramagnetic agent which

is used in clinical imaging to enhance tissue contrast and

to measure perfusion produces an increase in signal in-

tensity in T1-weighted images Analysis of these changes

in intensity can be used to produce concentration maps

of the contrast agent15 Thus by acquiring a series of im-

ages following injection of the contrast agent the per-

fusion of the scaffolds can be monitored This is illus-

trated in Fig 3 which shows a series of T1-weighted

images acquired from a scaffold after injection of the

contrast agent on days 7 and 14 after cell seeding The

increase in signal intensity approximately 6 min after

contrast agent injection indicated arrival of the agent in

the spaces around the scaffold There was then an in-

crease in signal within the scaffold as it became infil-

trated by the contrast agent The rate of contrast agent

NEVES ET AL

inflow into the scaffold was clearly faster on day 7 than

on day 14 The arrow in Fig 3A indicates a region that

enhanced relatively rapidly This contained a needle hole

that was made in the scaffold during cell seeding The

needle hole had become blocked by day 14 Figure 4

shows the time-dependent changes in contrast agent con-

centration in five different regions of interest (ROIs) in

the two different scaffolds perfused at different flow rates

(30 and 60 mL min21) Four of these ROIs were within

the volume of the construct (ROI1 to ROI4 labeled in

the direction of medium flow) and the fifth was imme-

diately above it These perfusion profiles were obtained

immediately after cell seeding and on days 7 and 14 of

cultivation The perfusion profiles obtained immediately

after cell seeding of the four internal ROIs and the ex-

ternal ROI were similar to each other and appeared to be

independent of the flow rate The similarity of the pro-

files for the external and internal ROIs coupled with the

direct measurements of linear flow velocities within the

construct (see below) showed that penetration of the con-

54

FIG 2 (A) Diffusion-weighted MR images acquired from a cell-seeded scaffold during a bioreactor run The image plane was

within the construct and was parallel to the direction of flow Signal intensity is inversely proportional to the ADC of water that

is high signal intensity corresponds to a higher cell content (see text) (i) Flow rate 5 30 mL min21 (ii) flow rate 5 60 mL

min21 (B) (i) Relative time-dependent changes in the average signal intensity in the diffusion-weighted images shown in (A)

and (ii) the NTP content of the constructs determined by 31P MRS (s) Flow rate 5 30 mL min21 (j) flow rate 5 60 mL min21

trast agent into the scaffold at this stage of the culture

was driven primarily by flow rather than by diffusion

However by day 7 in the scaffold perfused at a flow rate

of 30 mL min21 the rate of inflow of contrast agent was

much reduced and this was further reduced by day 14

Closer examination of the perfusion profiles obtained on

days 7 and 14 shows that the regions exposed directly

(ROI1) or indirectly (ROI4) to the flow were now more

efficiently perfused than the internal ROIs This indicates

that diffusion rather than flow is now primarily respon-

sible for inflow of the contrast agent into the construct

The diffusion coefficient for the contrast agent in the

outer ROIs was determined by using a simple one-di-

mensional non-steady-state diffusion model (infinite slab

model) proposed by Crank22 (see Fig 5A) We have used

this model previously to determine the diffusion coeffi-

cient of Gd-DTPA in cell-filled carriers in a fixed-bed

bioreactor15 A diffusion coefficient of (7 6 05) 3

10211 m2 s21 was determined for ROI1 which is simi-

lar to a value of 92 3 10211 m2 s21 found by Foy and

Blake23 for the diffusion of Gd-DTPA in native human

articular cartilage

The value of R1p which is directly proportional to the

concentration of the contrast agent can be used to esti-

mate the volume fraction of the construct from which the

agent is excluded15 By day 14 at a flow rate of 30 mL

MRI ANALYSIS OF MENISCAL CARTILAGE

min21 the excluded volume fraction was estimated at

55 (Fig 5B) Removal of the construct at this stage and

subsequent cell counting gave a cell volume fraction of

53 assuming that a fibrochondrocyte has a volume of

1028 cm3 (estimated by using a calibration of cell num-

ber vs wet weight results not shown) The collagen and

GAG contents were measured at 57 and 11 of the fi-

nal dry weight respectively (or 06 and 011 of the

final wet weight) and thus could make only a negligible

contribution to the excluded volume fraction Measure-

ments of NTP content and cell counting indicated that at

this flow rate the cell number in the scaffold increased

by nearly a factor of 2 during the 14 days of the culture

Yet the excluded volume data suggested that there was a

nearly 7-fold increase in cell content from 8 on day 1

to 55 on day 14 This underestimate of the excluded or

cell volume fraction on day 1 is due to the effects of flow

which by decreasing the apparent T1 increased the esti-

mated R1p or Gd-DTPA concentration in the scaffold

However by day 14 the increase in cell number and de-

position of matrix material has reduced flow through the

construct to undetectable levels (see below) and thus the

measured water T1 in the construct is now a true reflec-

tion of the Gd-DTPA concentration This also explains

why at a flow rate of 60 mL min21 at which there is al-

ways macroscopic flow of material through the construct

55

FIG 3 Time course of Gd-DTPA inflow in a construct on day 7 (A) and day 14 (B) of the bioreactor run The imaging plane

was parallel to the direction of flow and perpendicular to the plane of the construct The medium flow rate was 30 mL min21

The arrow indicates the location of the hole made in the scaffold during the seeding procedure The numbers indicate the time

(minutes seconds) after injection of the contrast agent into the medium conditioning vessel (see Materials and Methods)

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

contrast agent inflow into the constructs was observed

with a series of T1-weighted spin-echo images The ac-

quisition parameters for these images were TR 5 130 ms

TE 5 123 ms and the slice thickness was 20 mm The

field-of-view was 20 3 20 mm acquired into 512 3 128

(phase encode) data points giving an in-plane resolution

of 008 3 016 mm Maps of the paramagnetic contribu-

tion to the relaxation rate (R1p) were derived for each of

the T1-weighted images in the time course1115 R1p is di-

rectly proportional to the concentration of the contrast

agent

MRI measurements of flow

Axial flow through and around the scaffolds was mea-

sured by a time-of-flight MR imaging method This was

based on a selective inversion recovery pulse sequence in

which slice-selective spin-tagging and detection pulses

were followed by a bipolar readout gradient18 Flow rates

were determined on a pixel-by-pixel basis from the flow-

dependent changes in the apparent T1 The acquisition

parameters for these images were TR 5 01 ms TE 5

273 ms and the slice thickness was 30 mm Images were

acquired at 12 different delays ranging from 000625 to

64 s between the 180deg slice-selective inversion pulse and

the low flip-angle slice-selective detection pulse This

range of delays was chosen to ensure full recovery of the

water proton magnetization to its equilibrium state both

in the presence and absence of flow The field-of-view

was 25 3 25 mm acquired into 64 3 64 (phase encode)

data points giving an in-plane resolution of 02 3

MRI ANALYSIS OF MENISCAL CARTILAGE

04 mm Four transients were acquired per phase encode

increment giving a total image acquisition time of 40 s

31P magnetic resonance spectroscopymeasurements of cellular energy metabolism

31P nuclear magnetic resonance (NMR) spectra of the

bioreactors were acquired as described previously11 A

40-ms 90deg pulse and a repetition time of 14 s were used

Histologic analysis

Constructs were harvested from the bioreactors at the

end of the cultivation period of 2 weeks and immersed

in a 4 solution of formaldehyde before histologic ex-

amination Samples were later dehydrated with graded

concentrations of ethanol and embedded in glycol

methacrylate (GMA) resin using an embedding kit

(Technovit 7100 TAA Laboratories Equipment Alder-

maston UK) Blocks were sectioned and histologic sec-

tions (8 mm thick) were produced by an automated mi-

crotome with disposable tungsten carbide knives

Sections were stained with Mayerrsquos hematoxylin and

phloxine B (Sigma Dorset UK)

Collagen and glycosaminoglycan analysis

The glycosaminoglycan (GAG) content of the samples

was determined spectrophotometrically using the di-

methylmethylene blue dye (DMB) method19 Total col-

lagen content was determined from the measured hy-

droxyproline content of the constructs after acid

hydrolysis and reaction with p-dimethylaminobenzalde-

hyde and chloramine-T20 using a ratio of hydroxypro-

line to collagen of 014321

RESULTS

Cell distribution and content

The time-dependent changes in the signal intensities

in diffusion-weighted images of scaffolds perfused at two

different flow rates are shown in Fig 2 These changes

parallel to some extent changes in the nucleoside

triphosphate (NTP) content of the scaffolds determined

by 31P MRS (Fig 2B) as has been observed previously

for CHO cells growing in a fixed-bed bioreactor11 and is

consistent with signal intensity in the diffusion-weighted

MR image being primarily a measure of cell content

Both sets of data indicate that at 30 mL min21 there was

a progressive increase in cell content over the 2 weeks

of culture However at 60 mL min21 there was an ini-

tial increase in cell content followed by a subsequent de-

cline in the second half of the culture which was ob-

served in the diffusion-weighted images as a loss of signal

intensity at the center of the scaffold (Fig 2A)

53

The T1 or spin-lattice relaxation time is the time constant

for recovery of the bulk magnetization in an NMR experiment

to its equilibrium value following a perturbation The relaxation

rate (R1) is the inverse of this (R1 5 1T1) In the T1-weighted

imaging experiment used here signal intensity is directly pro-

portional to the relaxation rate This in turn is directly pro-

portional to the concentration of the contrast agent Gd-DTPA

which enhances spin-lattice relaxation

FIG 1 Bioreactor design (A) Flow cell with cell scaffold

(indicated by cross-hatching) located between two supporting

disks (B) Top view of a supporting disk The slots around the

edge allow for diversion of flow away from the construct as

this becomes progressively blocked with cells and extracellu-

lar matrix material

Scaffold perfusion

Perfusion of the scaffolds was measured by adding

a contrast agent (Gd-DTPA molecular weight 590

gmol21) to the medium This paramagnetic agent which

is used in clinical imaging to enhance tissue contrast and

to measure perfusion produces an increase in signal in-

tensity in T1-weighted images Analysis of these changes

in intensity can be used to produce concentration maps

of the contrast agent15 Thus by acquiring a series of im-

ages following injection of the contrast agent the per-

fusion of the scaffolds can be monitored This is illus-

trated in Fig 3 which shows a series of T1-weighted

images acquired from a scaffold after injection of the

contrast agent on days 7 and 14 after cell seeding The

increase in signal intensity approximately 6 min after

contrast agent injection indicated arrival of the agent in

the spaces around the scaffold There was then an in-

crease in signal within the scaffold as it became infil-

trated by the contrast agent The rate of contrast agent

NEVES ET AL

inflow into the scaffold was clearly faster on day 7 than

on day 14 The arrow in Fig 3A indicates a region that

enhanced relatively rapidly This contained a needle hole

that was made in the scaffold during cell seeding The

needle hole had become blocked by day 14 Figure 4

shows the time-dependent changes in contrast agent con-

centration in five different regions of interest (ROIs) in

the two different scaffolds perfused at different flow rates

(30 and 60 mL min21) Four of these ROIs were within

the volume of the construct (ROI1 to ROI4 labeled in

the direction of medium flow) and the fifth was imme-

diately above it These perfusion profiles were obtained

immediately after cell seeding and on days 7 and 14 of

cultivation The perfusion profiles obtained immediately

after cell seeding of the four internal ROIs and the ex-

ternal ROI were similar to each other and appeared to be

independent of the flow rate The similarity of the pro-

files for the external and internal ROIs coupled with the

direct measurements of linear flow velocities within the

construct (see below) showed that penetration of the con-

54

FIG 2 (A) Diffusion-weighted MR images acquired from a cell-seeded scaffold during a bioreactor run The image plane was

within the construct and was parallel to the direction of flow Signal intensity is inversely proportional to the ADC of water that

is high signal intensity corresponds to a higher cell content (see text) (i) Flow rate 5 30 mL min21 (ii) flow rate 5 60 mL

min21 (B) (i) Relative time-dependent changes in the average signal intensity in the diffusion-weighted images shown in (A)

and (ii) the NTP content of the constructs determined by 31P MRS (s) Flow rate 5 30 mL min21 (j) flow rate 5 60 mL min21

trast agent into the scaffold at this stage of the culture

was driven primarily by flow rather than by diffusion

However by day 7 in the scaffold perfused at a flow rate

of 30 mL min21 the rate of inflow of contrast agent was

much reduced and this was further reduced by day 14

Closer examination of the perfusion profiles obtained on

days 7 and 14 shows that the regions exposed directly

(ROI1) or indirectly (ROI4) to the flow were now more

efficiently perfused than the internal ROIs This indicates

that diffusion rather than flow is now primarily respon-

sible for inflow of the contrast agent into the construct

The diffusion coefficient for the contrast agent in the

outer ROIs was determined by using a simple one-di-

mensional non-steady-state diffusion model (infinite slab

model) proposed by Crank22 (see Fig 5A) We have used

this model previously to determine the diffusion coeffi-

cient of Gd-DTPA in cell-filled carriers in a fixed-bed

bioreactor15 A diffusion coefficient of (7 6 05) 3

10211 m2 s21 was determined for ROI1 which is simi-

lar to a value of 92 3 10211 m2 s21 found by Foy and

Blake23 for the diffusion of Gd-DTPA in native human

articular cartilage

The value of R1p which is directly proportional to the

concentration of the contrast agent can be used to esti-

mate the volume fraction of the construct from which the

agent is excluded15 By day 14 at a flow rate of 30 mL

MRI ANALYSIS OF MENISCAL CARTILAGE

min21 the excluded volume fraction was estimated at

55 (Fig 5B) Removal of the construct at this stage and

subsequent cell counting gave a cell volume fraction of

53 assuming that a fibrochondrocyte has a volume of

1028 cm3 (estimated by using a calibration of cell num-

ber vs wet weight results not shown) The collagen and

GAG contents were measured at 57 and 11 of the fi-

nal dry weight respectively (or 06 and 011 of the

final wet weight) and thus could make only a negligible

contribution to the excluded volume fraction Measure-

ments of NTP content and cell counting indicated that at

this flow rate the cell number in the scaffold increased

by nearly a factor of 2 during the 14 days of the culture

Yet the excluded volume data suggested that there was a

nearly 7-fold increase in cell content from 8 on day 1

to 55 on day 14 This underestimate of the excluded or

cell volume fraction on day 1 is due to the effects of flow

which by decreasing the apparent T1 increased the esti-

mated R1p or Gd-DTPA concentration in the scaffold

However by day 14 the increase in cell number and de-

position of matrix material has reduced flow through the

construct to undetectable levels (see below) and thus the

measured water T1 in the construct is now a true reflec-

tion of the Gd-DTPA concentration This also explains

why at a flow rate of 60 mL min21 at which there is al-

ways macroscopic flow of material through the construct

55

FIG 3 Time course of Gd-DTPA inflow in a construct on day 7 (A) and day 14 (B) of the bioreactor run The imaging plane

was parallel to the direction of flow and perpendicular to the plane of the construct The medium flow rate was 30 mL min21

The arrow indicates the location of the hole made in the scaffold during the seeding procedure The numbers indicate the time

(minutes seconds) after injection of the contrast agent into the medium conditioning vessel (see Materials and Methods)

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

Scaffold perfusion

Perfusion of the scaffolds was measured by adding

a contrast agent (Gd-DTPA molecular weight 590

gmol21) to the medium This paramagnetic agent which

is used in clinical imaging to enhance tissue contrast and

to measure perfusion produces an increase in signal in-

tensity in T1-weighted images Analysis of these changes

in intensity can be used to produce concentration maps

of the contrast agent15 Thus by acquiring a series of im-

ages following injection of the contrast agent the per-

fusion of the scaffolds can be monitored This is illus-

trated in Fig 3 which shows a series of T1-weighted

images acquired from a scaffold after injection of the

contrast agent on days 7 and 14 after cell seeding The

increase in signal intensity approximately 6 min after

contrast agent injection indicated arrival of the agent in

the spaces around the scaffold There was then an in-

crease in signal within the scaffold as it became infil-

trated by the contrast agent The rate of contrast agent

NEVES ET AL

inflow into the scaffold was clearly faster on day 7 than

on day 14 The arrow in Fig 3A indicates a region that

enhanced relatively rapidly This contained a needle hole

that was made in the scaffold during cell seeding The

needle hole had become blocked by day 14 Figure 4

shows the time-dependent changes in contrast agent con-

centration in five different regions of interest (ROIs) in

the two different scaffolds perfused at different flow rates

(30 and 60 mL min21) Four of these ROIs were within

the volume of the construct (ROI1 to ROI4 labeled in

the direction of medium flow) and the fifth was imme-

diately above it These perfusion profiles were obtained

immediately after cell seeding and on days 7 and 14 of

cultivation The perfusion profiles obtained immediately

after cell seeding of the four internal ROIs and the ex-

ternal ROI were similar to each other and appeared to be

independent of the flow rate The similarity of the pro-

files for the external and internal ROIs coupled with the

direct measurements of linear flow velocities within the

construct (see below) showed that penetration of the con-

54

FIG 2 (A) Diffusion-weighted MR images acquired from a cell-seeded scaffold during a bioreactor run The image plane was

within the construct and was parallel to the direction of flow Signal intensity is inversely proportional to the ADC of water that

is high signal intensity corresponds to a higher cell content (see text) (i) Flow rate 5 30 mL min21 (ii) flow rate 5 60 mL

min21 (B) (i) Relative time-dependent changes in the average signal intensity in the diffusion-weighted images shown in (A)

and (ii) the NTP content of the constructs determined by 31P MRS (s) Flow rate 5 30 mL min21 (j) flow rate 5 60 mL min21

trast agent into the scaffold at this stage of the culture

was driven primarily by flow rather than by diffusion

However by day 7 in the scaffold perfused at a flow rate

of 30 mL min21 the rate of inflow of contrast agent was

much reduced and this was further reduced by day 14

Closer examination of the perfusion profiles obtained on

days 7 and 14 shows that the regions exposed directly

(ROI1) or indirectly (ROI4) to the flow were now more

efficiently perfused than the internal ROIs This indicates

that diffusion rather than flow is now primarily respon-

sible for inflow of the contrast agent into the construct

The diffusion coefficient for the contrast agent in the

outer ROIs was determined by using a simple one-di-

mensional non-steady-state diffusion model (infinite slab

model) proposed by Crank22 (see Fig 5A) We have used

this model previously to determine the diffusion coeffi-

cient of Gd-DTPA in cell-filled carriers in a fixed-bed

bioreactor15 A diffusion coefficient of (7 6 05) 3

10211 m2 s21 was determined for ROI1 which is simi-

lar to a value of 92 3 10211 m2 s21 found by Foy and

Blake23 for the diffusion of Gd-DTPA in native human

articular cartilage

The value of R1p which is directly proportional to the

concentration of the contrast agent can be used to esti-

mate the volume fraction of the construct from which the

agent is excluded15 By day 14 at a flow rate of 30 mL

MRI ANALYSIS OF MENISCAL CARTILAGE

min21 the excluded volume fraction was estimated at

55 (Fig 5B) Removal of the construct at this stage and

subsequent cell counting gave a cell volume fraction of

53 assuming that a fibrochondrocyte has a volume of

1028 cm3 (estimated by using a calibration of cell num-

ber vs wet weight results not shown) The collagen and

GAG contents were measured at 57 and 11 of the fi-

nal dry weight respectively (or 06 and 011 of the

final wet weight) and thus could make only a negligible

contribution to the excluded volume fraction Measure-

ments of NTP content and cell counting indicated that at

this flow rate the cell number in the scaffold increased

by nearly a factor of 2 during the 14 days of the culture

Yet the excluded volume data suggested that there was a

nearly 7-fold increase in cell content from 8 on day 1

to 55 on day 14 This underestimate of the excluded or

cell volume fraction on day 1 is due to the effects of flow

which by decreasing the apparent T1 increased the esti-

mated R1p or Gd-DTPA concentration in the scaffold

However by day 14 the increase in cell number and de-

position of matrix material has reduced flow through the

construct to undetectable levels (see below) and thus the

measured water T1 in the construct is now a true reflec-

tion of the Gd-DTPA concentration This also explains

why at a flow rate of 60 mL min21 at which there is al-

ways macroscopic flow of material through the construct

55

FIG 3 Time course of Gd-DTPA inflow in a construct on day 7 (A) and day 14 (B) of the bioreactor run The imaging plane

was parallel to the direction of flow and perpendicular to the plane of the construct The medium flow rate was 30 mL min21

The arrow indicates the location of the hole made in the scaffold during the seeding procedure The numbers indicate the time

(minutes seconds) after injection of the contrast agent into the medium conditioning vessel (see Materials and Methods)

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

trast agent into the scaffold at this stage of the culture

was driven primarily by flow rather than by diffusion

However by day 7 in the scaffold perfused at a flow rate

of 30 mL min21 the rate of inflow of contrast agent was

much reduced and this was further reduced by day 14

Closer examination of the perfusion profiles obtained on

days 7 and 14 shows that the regions exposed directly

(ROI1) or indirectly (ROI4) to the flow were now more

efficiently perfused than the internal ROIs This indicates

that diffusion rather than flow is now primarily respon-

sible for inflow of the contrast agent into the construct

The diffusion coefficient for the contrast agent in the

outer ROIs was determined by using a simple one-di-

mensional non-steady-state diffusion model (infinite slab

model) proposed by Crank22 (see Fig 5A) We have used

this model previously to determine the diffusion coeffi-

cient of Gd-DTPA in cell-filled carriers in a fixed-bed

bioreactor15 A diffusion coefficient of (7 6 05) 3

10211 m2 s21 was determined for ROI1 which is simi-

lar to a value of 92 3 10211 m2 s21 found by Foy and

Blake23 for the diffusion of Gd-DTPA in native human

articular cartilage

The value of R1p which is directly proportional to the

concentration of the contrast agent can be used to esti-

mate the volume fraction of the construct from which the

agent is excluded15 By day 14 at a flow rate of 30 mL

MRI ANALYSIS OF MENISCAL CARTILAGE

min21 the excluded volume fraction was estimated at

55 (Fig 5B) Removal of the construct at this stage and

subsequent cell counting gave a cell volume fraction of

53 assuming that a fibrochondrocyte has a volume of

1028 cm3 (estimated by using a calibration of cell num-

ber vs wet weight results not shown) The collagen and

GAG contents were measured at 57 and 11 of the fi-

nal dry weight respectively (or 06 and 011 of the

final wet weight) and thus could make only a negligible

contribution to the excluded volume fraction Measure-

ments of NTP content and cell counting indicated that at

this flow rate the cell number in the scaffold increased

by nearly a factor of 2 during the 14 days of the culture

Yet the excluded volume data suggested that there was a

nearly 7-fold increase in cell content from 8 on day 1

to 55 on day 14 This underestimate of the excluded or

cell volume fraction on day 1 is due to the effects of flow

which by decreasing the apparent T1 increased the esti-

mated R1p or Gd-DTPA concentration in the scaffold

However by day 14 the increase in cell number and de-

position of matrix material has reduced flow through the

construct to undetectable levels (see below) and thus the

measured water T1 in the construct is now a true reflec-

tion of the Gd-DTPA concentration This also explains

why at a flow rate of 60 mL min21 at which there is al-

ways macroscopic flow of material through the construct

55

FIG 3 Time course of Gd-DTPA inflow in a construct on day 7 (A) and day 14 (B) of the bioreactor run The imaging plane

was parallel to the direction of flow and perpendicular to the plane of the construct The medium flow rate was 30 mL min21

The arrow indicates the location of the hole made in the scaffold during the seeding procedure The numbers indicate the time

(minutes seconds) after injection of the contrast agent into the medium conditioning vessel (see Materials and Methods)

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

(see below) the estimated volume fraction was always

lower than the real fraction based on cell counting and

analysis of collagen and GAG content Thus on day 14

at this higher flow rate the total excluded volume frac-

tion based on cell counting and analysis of extracellular

matrix materials was 34 whereas the estimate based

on measurement of contrast agent concentration was

only 15

Flow

Axial velocity profiles for different cross sections of the

bioreactor were determined using a method based on the

principle of time-of-flight18 The T1 map produced by this

imaging sequence when appropriately calibrated can be

used to produce a map of the axial flow velocities in the

construct and surrounding bioreactor Such a calibration is

shown in Fig 6 for a phantom consisting of two coaxial

tubes This geometry was chosen because it is similar to

that of the bioreactor and produces a laminar flow profile

that can easily be calculated using fluid dynamics theory

Flow velocities in the central tube showed a parabolic pro-

file that is typical of laminar flow in a cylinder (Fig 6A)

There was good agreement between the observed veloci-

ties and those expected from theory (Fig 6B) In addition

NEVES ET AL

laminar flow in the annular region of the phantom produced

the expected semitorispherical flow profile The calibration

depends on the true T1 of the medium that is the T1 in the

absence of flow and therefore was repeated at different Gd-

DTPA concentrations so that these flow measurements

could be interleaved with the measurements of construct

perfusion using the contrast agent There was a linear rela-

tionship between the flow-dependent changes in the ap-

parent T1 relaxation time and the Gd-DTPA concentration

(data not shown)

The flow velocities in an unseeded scaffold showed

the expected linear relationship with medium flow rate

(Fig 7AndashD) The flow rate (F) in a cylinder is defined

by fluid flow theory as the product of the average ve-

locity of the liquid (v) and the area of the cylinderrsquos cross-

section (A) A linear relationship between F and v indi-

cates therefore a constant A which in this case is the void

area inside the scaffold through which flow occurs These

velocities were reduced after cell seeding (Fig 7E and

F) The marked decrease in flow velocities between day

1 and day 14 for the scaffold perfused at 30 mL min21

and the smaller decreases observed in the scaffold per-

fused at 60 mL min21 are consistent with the cell den-

sities measured directly by cell counting and those in-

ferred from 31P MRS measurements of NTP content (Fig

56

FIG 4 Time-dependent changes in contrast agent concentration (expressed as the paramagnetic contribution to relaxation rate

R1p) in specific regions of interest (ROIs) within (ROI1 to ROI4) and immediately above the construct (ROI5) The ROIs are la-

beled in the direction of the flow (ascending) The location of the ROIs which bisected the center of the construct and measured

3 3 95 3 085 mm is indicated in (C) Perfusion profiles for two constructs perfused at two different flow rates 30 mL min21

(A) and 60 mL min21 (B) are shown Key (m) ROI1 (r) ROI2 (j) ROI3 (u) ROI4 (d) ROI5

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

2B) from diffusion-weighted MRI measurements (Fig

2A and B) and from contrast agent-enhanced MRI mea-

surements of the excluded volume fraction (Fig 5B) The

initial velocity profiles obtained on day 1 from the cell-

seeded scaffolds (Fig 7E and F) show greater flow ve-

locities at the center of the scaffolds because of the hole

made during the cell-seeding process This hole (ap-

proximately 07 mm in diameter) was completely blocked

after 7 days of cell culture at a flow rate of 30 mL min21

but did not become blocked even after 14 days of cul-

ture at 60 mL min21 The higher flow rate clearly pro-

duced better perfusion of the scaffold although ulti-

mately this was destructive reducing the cell and matrix

content at the center of the scaffolds This was observed

in the diffusion-weighted imaging (Fig 2) and was con-

firmed by subsequent histologic analysis (Fig 8)

The constructs showed a distinctive morphology which

was most clearly observed at a flow rate of 30 mL min21

Cells located at the periphery were aligned perpendicularly

to the flow of medium and exhibited a fibroblast-like mor-

phology The formation of this external layer has been re-

ported previously for bioartificial cartilage cultured in spin-

ner flasks17 and in bioreactors8 The cells in the core of

the constructs displayed a morphology that more closely

resembled that of fibrochondrocytes in native meniscal tis-

sue that is rounded cells that had larger spaces between

them Nevertheless some of these cells had pyknotic nu-

clei indicating possible nutrient deprivation

DISCUSSION

Perfusion bioreactor systems have the advantage that

in principle they allow close control of cell culture con-

MRI ANALYSIS OF MENISCAL CARTILAGE

ditions However their use as a system to generate fi-

brocartilaginous tissues with properties suitable for

transplantation requires the specification of a range of

operating parameters These include cell-seeding density

medium selection (which might include specific growth

factor supplementation24) selection of appropriate ma-

trices on which to grow the cells4 and the application of

physical stimuli such as pressure and flow-induced shear

stress that have been shown to influence cell growth and

extracellular matrix composition25 In the case of menis-

cal cartilage tissue limited experience with the in vitro

culture of meniscal fibrochondrocytes has provided some

information26 However the optimal conditions under

which meniscal cartilage can be grown in vitro with com-

position and properties most closely resembling those

found in vivo have yet to be determined6

MRI has been used predominantly as a technique for

imaging tissue anatomy in the clinic However numer-

ous MRI methods have been developed that report on

other aspects of tissue physiology27 including brain func-

tion arterial blood flow and tissue perfusion Some of

these methods could potentially be useful for character-

izing the behavior of engineered tissues and indeed there

are now many examples of MR-based methods that have

been used to assess the performance of mammalian cell

bioreactor systems of the type that could be used for tis-

sue-engineering applications14

The goal of the present study was to validate MR-based

methods for optimizing the operating conditions of a tis-

sue-engineering bioreactor in order to generate meniscal

cartilage constructs with properties approaching those of

the native tissue Diffusion-weighted MRI experiments

and 31P MRS measurements of NTP content were used

to monitor cell growth and distribution and MRI mea-

57

FIG 5 Inflow of Gd-DTPA into tissue-engineered meniscal cartilage constructs (A) The perfusion profile for ROI1 (see Fig

4) on day 14 of cultivation The contrast agent concentration is expressed as the paramagnetic contribution to the relaxation rate

(R1p) The data were fitted (solid line) to an infinite slab model in order to determine the diffusion coefficient of the contrast

agent in the construct (B) Excluded volume fraction in the construct as a function of the cultivation time and flow rate

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

NEVES ET AL58

FIG 6 Measurements of flow in a phantom The phantom consisted of two concentric tubes of diameter 64 and 207 mm

Water was pumped through the central tube at the specified flow rate and returned in the annular space between the two tubes

(A) Linear flow velocities (in mm s21) were estimated by the time-of-flight MRI method (see text) (B) The radial flow ve-

locity profile for a cross-section of the inner tube The flow rate was 40 mL min21 The data were fitted (solid line) to the

HaumlgenndashPoiseille equation V 5 Vmax [1 2 (rr0)2] where V(r) is the linear velocity at radius r and r0 is the radius of the in-

ner tube

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

MRI ANALYSIS OF MENISCAL CARTILAGE 59

FIG 7 Linear flow velocities in unseeded and cell-seeded scaffolds Maps of flow velocities were acquired from a 3-mm-thick

slice within the plane of the scaffolds that is perpendicular to the direction of medium flow (AndashD) Flow velocities at the spec-

ified flow rates in unseeded scaffolds (E and F) Flow velocities in cell-seeded scaffolds on the specified days of a bioreactor

run at the medium flow rates of 30 mL min21 (E) and 60 mL min21 (F)

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

surements were used to measure the bulk flow of medium

around and within the constructs and to measure their

perfusion by a small-molecule contrast agent The non-

invasive nature of these measurements allowed a con-

tinuous assessment of bioreactor behavior and the influ-

ence of different flow rates on its performance This

information would have been difficult to obtain by con-

ventional analytical methods For example histologic

analysis could have revealed cell distribution in the scaf-

folds but only at the end of a reactor run Laser Doppler

techniques could have been used to measure flow28 how-

ever the introduction of sensors into the reactor may

have affected reactor performance as could the intro-

duction of tracer particles28 Furthermore this technique

would not have been capable of monitoring flow within

the constructs themselves

A relatively high magnetic field was used in the ex-

periments described here 94 T when compared with the

fields commonly used in clinical MRI systems typically

between 15 and 3 T The greater sensitivity that results

from the use of a higher field strength allowed the ac-

quisition of images with much better spatial resolution

For example Stone et al29 used a clinical MRI system

at 15 T to determine the volumes of knee meniscal car-

tilage With a 16- to 18-cm field-of-view and a data ac-

quisition matrix of 256 3 256 data points they obtained

NEVES ET AL

a spatial resolution of approximately 07 3 07 mm The

spatial resolutions in the images acquired in this study

were considerably better This was important in view of

the small size of the constructs used (12 mm in diame-

ter and 4 mm thick)

In the tissue-engineered meniscal cartilage constructs

described here essentially two main processes occur si-

multaneously proliferation of the fibrochondrocytes and

biosynthesis of their extracellular matrix the major com-

ponents being collagen (type I) and GAG The scaffolds

on which the cells were grown in this study were non-

biodegradable and thus there could be no remodeling of

the tissue as a result of scaffold degradation The MR

measurements of cell content and distribution and the

measurements of medium flow and construct perfusion

showed that as the cell mass increased there was a pro-

gressive decrease in the porosity of the construct and in

the global mass transfer rate A similar decrease in mass

transfer was observed by Freed and co-workers who

measured the permeability of an articular cartilage con-

struct to glucose30 In a perfusion system such as that

used here the medium is continuously renewed in the

vicinity of the construct thus eliminating the presence

of external concentration gradients via flow-associated

mixing However the resistance to internal mass trans-

fer prevailed throughout the cultures and was not re-

60

FIG 8 Histologic sections of 2-week-old constructs grown at medium flow rates of 30 mL min21 (A) and 60 mL min21 (B)

Magnifications of the regions indicated in (A) are shown in (1) and (2)

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

REFERENCES

1 OrsquoDriscoll SW The healing and regeneration of articular

cartilage J Bone Joint Surg Am 80 1795 1998

2 Temenoff JS and Mikos AG Review Tissue engi-

neering for regeneration of articular cartilage Biomateri-

als 21 431 2000

3 Fairbank TJ Knee joint changes after meniscectomy J

Bone Joint Surg 30B 664 1948

4 Freed LE Vunjak-Novakovic G Biron RJ Eagles

DB Lesnoy DC Barlow SK and Langer R Biode-

gradable polymer scaffolds for tissue engineering Biotech-

nology (NY) 12 689 1994

5 Uchio Y Ochi M Matsusaki M Kurioka H and Kat-

sube K Human chondrocyte proliferation and matrix syn-

thesis cultured in Atelocollagen gel J Biomed Mater Res

50 138 2000

6 Sweigart MA and Athanasiou KA Toward tissue en-

gineering of the knee meniscus Tissue Eng 7 111 2001

7 Vunjak-Novakovic G Obradovic B Martin I Bursac

PM Langer R and Freed LE Dynamic cell seeding of

polymer scaffolds for cartilage tissue engineering Biotech-

nol Prog 14 193 1998

8 Sittinger M Schultz O Keyszer G Minuth WW and

Burmester GR Artificial tissues in perfusion culture Int

J Artif Organs 20 57 1997

9 Carver SE and Heath CA Influence of intermittent

pressure fluid flow and mixing on the regenerative prop-

erties of articular chondrocytes Biotechnol Bioeng 65274 1999

10 Callies R Jackson ME and Brindle KM Measure-

ments of the growth and distribution of mammalian cells

in a hollow-fiber bioreactor using nuclear magnetic reso-

nance imaging Biotechnology (NY) 12 75 1994

11 Thelwall PE and Brindle KM Analysis of CHO-K1 cell

growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging Cytotechnology 15 1999

12 Pfeuffer J Flogel U and Leibfritz D Monitoring of cell

volume and water exchange time in perfused cells by dif-

fusion-weighted 1H NMR spectroscopy NMR Biomed 1111 1998

13 Williams SNO Callies RM and Brindle KM Map-

ping of oxygen tension and cell distribution in a hollow-

fiber bioreactor using magnetic resonance imaging Bio-

technol Bioeng 58 56 1997

14 Brindle KM Investigating the performance of intensive

mammalian cell bioreactor systems using magnetic reso-

nance imaging and spectroscopy Biotechnol Genet Eng

Rev 15 499 1998

61

solved by increasing the medium flow rate which be-

came destructive above 60 mL min21 From the contrast

agent-based measurements of construct perfusion the

diffusion coefficient of the agent (Gd-DTPA) was esti-

mated at the periphery and found to be similar to that

measured for the same agent in native articular carti-

lage23 This provides a clear indication that there will be

nutritional limitation of cells at the center of the con-

struct a fact that was confirmed by subsequent histo-

logic analysis which showed cells with pyknotic nuclei

at the center of the constructs

These studies have demonstrated as has been observed

previously for articular cartilage16 that generation of

meniscal cartilage tissue is highly dependent on bioreac-

tor operating conditions The rapid decrease in mass

transport which accompanies construct maturation is a

particularly important problem to address especially if

thicker implants are required We have shown here how

noninvasive MR methods can be used online to deter-

mine how bioreactor operating strategies and matrix

geometries can influence construct perfusion and cell

growth and thus can be used to optimize these parame-

ters MRI methods have also been shown to be capable

of assessing quantitatively the composition of cartilage

extracellular matrix material both in vitro in a bioreac-

tor31 and in vivo32 Because MRI is commonly used in

the clinic to assess the recovery of the knee meniscus

from traumatic injury33 there is the possibility that in the

future methods similar to those described here could be

used to characterize the behavior of a bioartificial carti-

lage construct postimplantation albeit with lower spatial

resolution

CONCLUSIONS

The local changes in cell density perfusion and

medium flow velocities in meniscal cartilage constructs

have been evaluated by MRI- and MRS-based meth-

ods Histologic analysis of the resulting constructs has

been evaluated and related to fluid movement There

was a distinct outer layer which contained cells with

a fibroblastic morphology The inner core of the con-

structs showed a lower cell density and a cell mor-

phology that was more characteristic of native menis-

cal tissue Consistent with the MR measurements of

flow and perfusion cells in this region showed some

evidence of nutrient deprivation as indicated by the

presence of pyknotic nuclei Thus these scaffolds are

not able to support homogeneous cell growth and ex-

tracellular matrix production throughout the whole

thickness of the constructs The MR-based methods de-

scribed and implemented here could be used to screen

alternative scaffold geometries and bioreactor opera-

tion strategies

MRI ANALYSIS OF MENISCAL CARTILAGE

ACKNOWLEDGMENTS

The authors thank M Anderson R Turner S Russell

and E Robinson (SampN Group Research Centre) for sup-

port and D Reed for manufacturing the bioreactors used

in these studies AAN thanks FCT (Portugal) for his

scholarship (PRAXIS XXIBD 1951999) The authors

acknowledge Smith amp Nephew plc for this collaboration

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Address reprint requests to

Andre A Neves

Department of Biochemistry

University of Cambridge

80 Tennis Court Road

Cambridge CB2 1GA UK

E-mail atrmdn2molebiocamacuk

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