Mechanical loading regimes affect glucose

and lactate metabolism of chondrocytes.

R.A.A. PullensApril 2004

BMTE 04.17

Part 2 of MSc-thesis

Thesis committee:F.P.T. BaaijensC.C. van DonkelaarC.W.J. OomensJ.M.R.J. HuygheB.G. Sengers

Eindhoven University of TechnologyFaculty of Biomedical Engineering

Abstract

Normally, articular cartilage functions well over a lifetime, but traumatic injury or thedegenerative changes associated with osteoarthritis can result in significant erosion of thearticular layer. The poor intrinsic repair capacity of chondrocytes restricts the processof cartilage tissue formation and creates a demand for tissue engineered cartilage.

To cope with all the challenging demands of cartilage tissue engineering, control of thetissue engineering proces is needed. Hence, a sophisticated bioreactor is required. Thebioreactor, which is used in this study, can stimulate constructs using direct compressionand perfuse them for supply of nutrients and removal of waste products. Chowdhurryet al.4 showed that it is possible to modulate chondrocyte metabolism (e.g. prolifera-tion or extracellular matrix (ECM) synthesis) by mechanical stimulation. The largestdifferences were seen between 12 hours (I12) and 1.5 hours (I1.5) of intermittent loading.Now that it is possible to direct the cells to different metabolic processes, the next stepfor tissue engineering is to investigate a way to control these processes. It is known thatglucose can be used for energy supply or for glycosaminoglycan synthesis and that inthose processes the production of lactate is different. It is hypothesized that the energymetabolism is different when the cells are proliferating or synthesizing ECM.

Hence, the aim of this study is to stimulate chondrocyte/agarose constructs with thetwo intermittent loading regimes (I12, I1.5), in order to stimulate proliferation or ECMsynthesis, and analyse whether or not the glucose consumption and/or lactate produc-tion are different.

It is demonstrated the chondrocytes subjected to different dynamic loading regimeshave different rates of glucose consumption and lactate production. The I12 group con-sumed glucose and produced lactate at a lower rate, than the controls and the I1.5 group.It is shown that the use of the ratio of produced lactate over consumed glucose, indi-cates differences between controls and the I12 and I1.5 groups. In addition, differences inDNA content and GAG production between the I12 and I1.5 groups are found. Of majorimportance in the latter issue is the fact that apparently similar stimulation protocolsbetween the latter study and the study of Chowdhurry et al.4 show opposite effects inproliferation and ECM synthesis.

The combined results indicate that the energy metabolism is different when the cellsare either proliferating or synthesizing ECM. Measuring chondroycte metabolism is con-cluded to be a promising tool to monitor cartilage TE, although monitoring metabolismalone is insufficient for a control system.

2

Samenvatting

Normaal blijft kraakbeen een leven lang goed functioneren, maar beschadigingen of ve-randeringen geassocieerd met artrose kunnen leiden tot ernstige erosie van de kraakbeenlaag. Door de slechte herstel capaciteit van de chondrocyten zal er nauwelijks weefselformatie optreden en is een behoefte ontstaan voor tissue engineered kraakbeen.

Om goed om te kunnen gaan met alle uitdagende eisen van tissue engineered kraak-been, is het nodig controle uit te oefenen op het tissue engineering proces. Hiervoor iseen geavanceerde bioreactor nodig. De bioreactor die gebruikt is in deze studie, kan con-structs mechanisch stimuleren via directe compressie en kan medium door de constructsleiden voor de aanvoer van voedingsstoffen en de afvoer van afvalstoffen. Chowdhurryet al.4 hebben aangetoond dat het mogelijk is om het metabolisme van chondrocyten(proliferatie of extracellulaire matrix synthese) te veranderen door mechanische stimu-latie. De grootste verschillen zijn gevonden tussen 12 uur en 1.5 uur periodieke belasting.Wanneer er rekening wordt gehouden met het feit dat glucose ook gebruikt kan wordenvoor de energie huishouding en voor glycosaminoglycaan synthese dan is het de vraagof het energie metabolisme anders is tussen proliferatie of synthese van extracellulairematrix.

Het doel van deze studie is om chondrocyte/agarose constructs te belasten met tweeintermitterende belasting protocols om proliferatie of extracellulaire matrix synthese testimuleren en daarbij te analyseren of de glucose consumptie en lactate productie ver-schillend zijn.

Deze studie laat zien dat de glucose consumptie en lactate productie van chondrocytenverschillend zijn wanneer ze belast worden met verschillende dynamische belasting pro-tocols. De 12 uur gestimuleerde groep consumeerden glucose langzamer en produceerdelactate langzamer dan de controle groep en de 1.5 uur gestimuleerde groep. Het is verderaangetoond dat het gebruik van ratios van de hoeveelheid geproduceerde lactate over dehoeveelheid geconsumeerde glucose niet vanzelfsprekend is, maar dat ze wel verschillenaangeven tussen de controle en de gestimuleerde groepen. Tevens zijn er verschillengevonden in glycosaminoglycaan productie van de cellen en de DNA hoeveelheid in deconstructs. Van belang is dat de schijnbaar gelijke stimulatie protocollen van deze studieen de studie van Chowdhurry et al.4 tegenovergestelde effecten in proliferatie en extracel-lulaire matrix synthese laten zien. Het valt te concluderen dat het energie metabolismeverschillend is wanneer de cellen prolifereren of extracellulaire matrix maken. Het metenvan het energie metabolisme is dus een veelbelovend gereedschap voor het volgen van hetkraakbeen tissue engineering proces, hoewel het energie metabolisme alleen niet genoegis voor een controle systeem.

3

Contents

Abstract 2

Samenvatting 3

1 Introduction 6

1.1 Tissue engineering of articular cartilage . . . . . . . . . . . . . . . . . . . 6

1.2 Cartilage metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2.1 Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.2.2 Glycosaminoglycan synthesis . . . . . . . . . . . . . . . . . . . . . 9

1.3 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2 Materials and Methods 11

2.1 Preparation of chondrocyte/agarose constructs . . . . . . . . . . . . . . . 11

2.2 Bioreactor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.4 Medium and construct analysis . . . . . . . . . . . . . . . . . . . . . . . . 13

3 Results 15

3.1 Medium analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.2 Biochemical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.3 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4 Discussion 19

5 Future research and recommendations 22

4

Bibliography 26

A Microelectrodes 27

A.1 pH measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

A.2 O2 measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

B Protocols 32

B.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

B.2 Cell harvesting protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

B.3 Obtaining an agarose gel seeded with chondrocytes . . . . . . . . . . . . . 33

B.4 Protocol heat inactivation of fetal bovine serum . . . . . . . . . . . . . . . 34

B.5 Glucose assay protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

B.6 Lactate assay protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

B.7 GAG assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

B.8 DNA assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

C Computational analysis 40

5

Chapter 1

Introduction

1.1 Tissue engineering of articular cartilage

Articular cartilage is found in synovial joints and acts as a load-bearing cushion anddistributes the applied forces transmitted through the joint to the underlying subchon-dral bone.29,46 Normally, articular cartilage functions well over a lifetime, but traumaticinjury or the degenerative changes associated with osteoarthritis (OA) can result in sig-nificant erosion of the articular layer. This leads to joint pain and instability.13 The poorintrinsic repair capacity of chondrocytes restricts the process of cartilage tissue formationand creates a demand for tissue engineered cartilage.

Development of in vitro engineered cartilage has been attempted in the early days oftissue engineering, because of its simple structure. Articular cartilage is aneural, alym-phatic and avascular and its extracellular matrix (ECM) is produced and maintained byone type of specialized cells, chondrocytes. Because of the avascularity, cell nutrientslike oxygen (O2) and glucose have to diffuse from the synovial fluid to the cells. Wasteproducts, like lactate, are removed from the matrix by the reverse route. Gradients ofthese metabolites will arise across the tissue, resulting in lower nutrient concentrationsin the deep layers. The oxygen tension, for example, is found to be around 0.5-1%40

in those layers, compared to 4-10% in the synovial fluid.8 The lower O2 concentrationcauses the cells to consume glucose anaerobically. So, despite the relative simple struc-ture, cartilage is a dynamic tissue with characteristics that contradict this tissue as anideal model for tissue engineering. When using chondrocytes for tissue engineering, moredifficulties arise. First, chondrocytes have to be cultured in a 3D environment, a scaffold,otherwise they will loose their phenotype.1 Secondly, chondrocytes from different donorshave been reported to behave differently.21 Finally, when a tissue engineered constructis placed in situ, it has to bear great biomechanical loads.16

To cope with all these challenging demands, the circumstances for culturing cartilageneed great control. Hence, a sophisticated bioreactor is required. Such a bioreactor isbeing developed in the IMBIOTOR project. The aim of the project is to develop anIntelligent Mini Bioreactor to control the in vitro growth of tissue-engineered cartilage.The novelty or intelligence will be the online use of local and bulk changes in the con-struct, to change online the input parameters to influence the overall performance. Thebioreactor, which was developed, can stimulate constructs using direct compression and

6

perfuse them for supply of nutrients and removal of waste products.Mechanical loading has been demonstrated to be important for the normal mainte-

nance of articular cartilage.3,9, 45 Dynamic or cyclic loading conditions, correspondingto physiological loading, change biosynthetic activity in articular cartilage.3,15,37,38,41,42

Consequently, deformational loading applied on tissue engineered cartilage constructs iswidely studied,6,11,12,15,18,25–28,38,41,42 and the chondrocyte seeded 3D constructs arefound to respond similar to the loading as articular cartilage explants.2,5 One of themodel systems, involving chondrocytes isolated from articular cartilage and embeddedin agarose gel, has been used successfully in deformational loading studies by severalgroups.2,4, 11,17–19,25–28 Lee et al.17 have demonstrated that dynamic compressive straininfluenced proteoglycan (PG) synthesis and cell proliferation in a distinct and frequencydependent manner.

Recently, Chowdhury et al.4 demonstrated a temporal regulation of cell metabolismby chondrocytes subjected to different dynamic mechanical conditioning regimes. Fre-quent bursts of intermittent compression (1 Hz) for longer periods (12 hours) favouredPG synthesis, whereas shorter bursts of intermittent compression (1.5 hours) tended tofavour cell proliferation (figure 1.1), even though the total number of 1 Hz cycles wasidentical. The uncoupled nature of the metabolic response suggests that it is possibleto direct the cells either to proliferation or the ECM synthesis at any time point of theculture period, which is useful for tissue engineering.

Figure 1.1: Effects of intermittent compression on 35SO4 (A) and [3H]-Tdr incorporation (B),which are respectively measures for proteoglycan synthesis and proliferation.4

Now that it is possible to direct the cells to different metabolic processes (proliferationand ECM synthesis), the next step is to investigate a way to control these processesduring tissue engineering of cartilage. In the first period of this study a literature re-view35 has been written containing more information about articular cartilage and tissueengineering. From this review it is concluded that the energy metabolism of chondro-cytes can be used to distinguish between proliferation and ECM synthesis and that keyparameters of this metabolism can be used as feedback for control of cartilage tissueengineering.

7

1.2 Cartilage metabolism

1.2.1 Energy

Glucose is the major metabolic nutrient of most types of connective tissue cells39 (figure1.2). The main two pathways of glucose degradation are anaerobic and aerobic gly-colysis. In anaerobic glycolysis, glucose is degraded to pyruvate which is converted tolactate, a reaction sequence which does not consume O2. In aerobic glycolysis, glucoseis degraded to pyruvate, which is utilized by mitochondrial enzymes of the tricarboxylicacid (TCA) cycle to generate NADH (figure 1.2). In the process of oxidative phospho-rylation (OXPHOS), transfer of electrons from the NADH molecules and formation ofthe corresponding oxidized substrate provides the proton-motive force for the subsequentsynthesis of adenosine triphosphate (ATP) (figure 1.3). The efficiency of anaerobic gly-colysis (2 mol ATP per mol glucose) is much lower than that of aerobic glycolysis (36mol ATP per mol glucose).

CO2

NH3

NH3

α-ketoglutarate

malate

glutamine

glutamate

pyruvate lactate

glucose

glucose-6-P glucose-1-P

fructose-6-P

fructose-1,6-diP

glycosaminoglycansynthesis

glycogen

UDP-glucose

acetyl coenzyme A

by

amin

otra

nsf

eras

eTCA cycle

oxaloacetate

aspartate

Figure 1.2: Simplified overview of metabolism in cartilage.35

8

Articular cartilage is characterised by a meagre O2 consumption, which is only 2-5% ofO2 consumption in liver or kidney cells,44 and minimal release of CO2.

22 This meansthat the TCA cycle is active in cartilage, but much less than in other tissues. It isbelieved that as much as 80% of the glucose is metabolized to lactate by anaerobicglycolysis.21,34,39,43 Although the ATP formation by OXPHOS is small, the functionalintegrity of OXPHOS is reported to be required for basal intracellular generation of ATPand for collagen and PG synthesis.14 Furthermore, an increased role of OXPHOS inchondrocyte function under conditions of cartilage stress and increased energy demandsis suggested, for example during OA24 and cartilage repair.14

O2

H2OATP

ADPe.g. NADH2

e.g. NAD

Reduced coenzyme

Oxidised coenzyme

Oxidativephosphorylation

Figure 1.3: Oxidative phosphorylation.

In addition to glucose, glutamine also plays an essential role in the metabolism of a varietyof cell types.31,32 Studies with isolated chondrocytes and slices of cartilage indicate thatcartilage requires amino acids, e.g. glutamine, for growth and as an energy source.39

The major pathways for glutamine metabolism are shown in figure 1.2. It can be seenthat glutamine can be deaminated to glutamate and then to α-ketoglutarate for energysupply.

1.2.2 Glycosaminoglycan synthesis

Glucose and glutamine are not only used in the energy metabolism of chondrocytes,but are also used in the synthesis of glycosaminoglycans (GAGs).34,39 These GAGsare a main part of the ECM’s proteoglycans. One of the principal nucleotide sugarswhich is needed as a building block of the polysaccharides is UDP-glucose. UDP-glucoseis obtained from glucose or glycolytic intermediates by a pyrophosphorylase reaction39

(reaction 1.1):

UTP + glucose-1-P ↔ UDP-glucose + PPi (1.1)

Another key reaction in the GAG synthesis is the formation of glucosamine-6-phosphateby the action of an aminotransferase (reaction 1.2).39 Glucose is used in the formationof fructose-6-phosphate30 and glutamine is the amino donor in the reaction.39,44

fructose-6-P + glutamine → glucosamine-6-P + glutamate (1.2)

9

1.3 Aim

Chowdhurry et al.4 showed that it is possible to modulate chondrocyte metabolism (e.g.proliferation or ECM synthesis) by mechanical stimulation. The largest differences wereseen between 12 hours (I12) and 1.5 hours (I1.5) of intermittent loading. When it istaken into account that glucose can be used for energy supply or for GAG synthesis,the question rises if the energy metabolism is different when the cells are proliferatingor synthesizing ECM. Although it is believed that glucose, lactate, O2, CO2, glutamineand NH3 are all key parameters for characterization of the energy metabolism (figure1.2),33,35 the main focus of this study is on glucose consumption and lactate productionfor analyzing the metabolism. When all the glucose is anaerobically converted to lactatethe ratio of produced lactate over consumed glucose will be 2.

It is hypothesized that the ratio between those two processes is different when thecells either proliferate or synthesize ECM, because when a glucose molecule is consumedfor GAG synthesis, no lactate is formed. Hence, the aim of this study is to stimulatechondrocyte/agarose constructs with the two intermittent loading regimes (I12, I1.5)found by Chowdhurry et al.,4 in order to stimulate proliferation or ECM synthesis, andanalyse whether or not the glucose consumptions and/or lactate productions are different(figure 1.4).

?=

Metabolism

Mechanical stimulation

ECM synthesisProliferation

Figure 1.4: Mechanical stimulation and metabolism. With mechanical stimulation it is possibleto direct the chondrocytes to different metabolic activities (proliferation and ECMsynthesis).4 The question is whether or not the metabolism is different in thoseprocesses.

10

Chapter 2

Materials and Methods

2.1 Preparation of chondrocyte/agarose constructs

Tissue dissectionFeet from 3-6 months old pigs were obtained from a local abattoir within 2-3 hours ofslaughter. Articular chondrocytes were isolated from the talus-navicular and calcaneus-cuboideum surfaces of the ankle joint. The feet were cleaned and submerged in a extransoap solution for at least 30 minutes. The joint was then placed in a class I laminarflow hood. The joint was exposed and full depth cartilage was carefully scraped off andplaced in a 50 ml tube containing DMEM (Appendix B.1) with 1 mg ml−1 collagenase(Appendix B.2).

Tissue digestionThe dissected tissues were enzymatically digested at 37◦C in DMEM containing 1 mgml−1 collagenase in an incubator at 37◦C / 5% CO2 for approximately 18 hours. Afterincubation the digested tissue suspension was filtered to isolate the cells. This was doneunder aseptic conditions by filtering the digested tissue first through a coarse filter toremove undigested tissue and then through a 20 µm-pore cell strainer. The cells in thefiltrate were then washed three times with PBS by repeated centrifugation at 2000 rpmfor 7.5 minutes and resuspended in DMEM (Appendix B.2). The cells were then countedand prepared for culture.

Chondrocyte/agarose constructsChondrocytes were harvested from five porcine ankle joints. After digestion of the tissuethe cell suspension was added to an equal volume of molten 6% (w/v) agarose type VIIin PBS to yield a final cell concentration of 25·106 cells ml−1 in 3% (w/v) agarose (Ap-pendix B.3). The chondrocyte/agarose suspension was transferred into a sterile stainlesssteel mould, containing holes measuring 13 mm in diameter and 3 mm in height. Thechondrocyte/agarose suspension was allowed to gel at 4◦C for 20 min to yield cylindricalconstructs. The constructs were placed in 2 ml DMEM at 37◦C / 5% CO2 for 1 hour toequilibrate the glucose concentration in the construct. The constructs were then trans-ferred to the bioreactors. The remaining constructs were cut in half, of which one halfwas stored in a -20◦C freezer for biochemical analysis and the other half was stored in4% formaldehyde for histological evaluation.

11

2.2 Bioreactor system

The bioreactor which is used to apply dynamical compression to tissue engineered con-structs is developed within the IMBIOTOR project. The inner diameter of the bioreactoris 15 mm and the height is 30 mm. The bottom of the bioreactor is equipped with aporous glass filter, which makes it possible to perfuse the construct using a roller pumpconnected to the bioreactor. Extra connections on the bioreactor make it possible to alsopump the medium around the sample (figure 2.1). A reservoir can be used to increase theamount of medium. A magnet inside the bioreactor is used to compress the construct.This is done by placing a magnet under the bioreactor which pulls the stimulation mag-net down. A plastic ring with four pillars with a total height of 2.45 mm is placed at thebottom of the bioreactor. This ensures that the initial strain of the 3 mm high constructsis 15%. A thin-walled cylinder with a length of 12 mm is placed on top of the magnet toconstrain the movement of the magnet. This prevents destruction of the construct dueto high impact loading.

In this study it was desirable to see rapid changes in the metabolite concentrations ofthe medium, therefore a low amount of medium had to be used. The maximum amountof medium which could be added to the bioreactor alone was 2 ml, therefore it was chosento exclude the reservoir from the setup. Without the reservoir it was not necessary touse the roller pump, which was an advantage, because in the protocol of Chowdhurryet al.4 the medium also was not pumped around. The connections at the bioreactor(diameter=±1 mm), were used for oxygenation of the constructs by attaching pieces ofsilicon tube to them.

� � � � � � � �� � � � � � � �� � � � � � � �� � � � � � � �

� � � � � � � �� � � � � � � �� � � � � � � �� � � � � � � �

Construct

Magnet

Porous glass filter

Blocks

(a) (b)

Figure 2.1: Schematic overview (a) and image (b) of the IMBIOTOR.

2.3 Protocol

Each bioreactor was filled with two ml of DMEM. The bioreactor was placed eccentricallyon a lab stirrer to move the magnet in the bioreactor up and down. This way dynamiccompression was applied to the constructs. Intermittent compression was applied for 1.5

12

(I1.5) or 12 (I12) hours compression with equivalent unstrained periods for a total periodof 138 hours (figure 2.2) with a compressive strain amplitude of 15% at 1 Hz. Controlconstructs (Ctr) were maintained in an unstrained state in a 12-well culture plate. Allconstructs were incubated for 138 hours at 37◦C/5% CO2. At t=0, 16, 23, 40, 47, 64,71, 138 hours 100 µl medium samples were taken for analysis of the glucose and lactateconcentrations. At the end of the culture period, the constructs were removed from thebioreactors and cut in half for biochemical and histological analysis.

181512 21 240 3 6 9

strainedunstrained

I1.5

I12

Time (hours)

Figure 2.2: Schematic diagram illustrating 24 hours of the compression regimes performed onchondrocyte/agarose constructs.

2.4 Medium and construct analysis

Medium analysisThe glucose and lactate concentration of the medium samples were determined usingthe Glucose Assay Kit (GAGO-20) from Sigma and the Lactate Assay kit (TB-735-10)from Kordia Life Sciences, respectively. Both assays were scaled for microplates andare based on measuring the absorbance of the samples at 540 nm using a plate reader.D(+)-Glucose (Merck) and L-(+)-Lactic Acid (Sigma) were used as standards for theassays (Appendices B.5 and B.6).

Biochemical analysisThe construct samples were weighted wet, lysophilized and reweighted dry. For thedetermination of GAG content per mg dry weight, the samples were digested with papainat 60◦C for 16 h. After the digestion, the samples were assessed using the DMMBassay,7 scaled for microplates. Chondroitin sulfate from bovine trachea (Sigma) wasused as standard (Appendix B.7). A GenElute Mammalian Genomic DNA MiniprepKit (G1N70, Sigma) was used to determine the amount of DNA per mg dry weight(Appendix B.8).

HistologyThe construct samples for histology were placed in formaldehyde for a minimum of24 hours and were then fixed overnight in xylene, and dehydrated in a graded seriesof ethanol. Samples were then embedded in paraffin, sectioned to 5 µm, and affixedto microscope slides. Sections were stained with hematoxylin/eosin to visualize thedistribution of the cells and neo formation of tissue. Stained sections were photographed(20x objective) using a digital camera.

13

Data analysis and statisticsThe 100 µl samples were a substantial amount of the total medium volume, thereforethe glucose and lactate concentrations could not be used directly for comparison ofthe consumption and production rates. Therefore, the measured concentrations weremultiplied with the volume of medium at the time of sampling. This gave the totalamount (µmol) of glucose and lactate present in the medium. Values are presentedas means ± standard deviation. The differences between the Ctr, I12 and I1.5 groupswere assessed by one way ANOVA. To make a comparison between the three groups alinear regression model was fitted to the linear part of the data and afterwards a GeneralLinear Model Repeated Measures Analysis was used to determine whether the slopes ofthe glucose consumptions and lactate productions were different. In all cases p<0.05 wasconsidered significant.

14

Chapter 3

Results

3.1 Medium analysis

The glucose consumption and the lactate production of the Ctr, I12 and I1.5 groups areshown in figures 3.1(a) and 3.1(b). It can be seen that the lactate production in the first16 hours was high, compared to the amount of consumed glucose. At t=71 hours almostall glucose was consumed, which caused the glucose consumption and lactate productionto level of from t=71 hours to t=138 hours. To compare the consumption and productionrates among the three groups, a linear regression model was fitted to the data points fromt=16 till t=71 hours (figures 3.1(a) and 3.1(b)). The matching equations and correlationcoefficients (R2) are summarized in table 3.1. It can be seen that the fits of the glucoseconsumptions are better than those of the lactate productions.

0 20 40 60 80 100 120 140

0

2

4

6

8

10

Time (hours)

Am

ount

of c

onsu

med

glu

cose

(µm

ol)

CtrI12I1.5Linear CtrLinear I12Linear I1.5

(a) Glucose consumption

0 20 40 60 80 100 120 140

0

2

4

6

8

10

12

14

16

18

20

Time (hours)

Am

ount

of p

rodu

ced

lact

ate

(µm

ol)

CtrI12I1.5Linear CtrLinear I12Linear I1.5

(b) Lactate production

Figure 3.1: Glucose consumption (a) and lactate production (b) from the Ctr, I12 and I1.5groups. Linear regression curves based on the data points from t=16 till t=71hours for each group are shown.

15

It can be seen that the final amount of consumed glucose (t=138 hours) differs betweenthe three groups. The I12 group consumed less glucose than the Ctr group and the I1.5group consumed more glucose (figure 3.1(a)). The I12 group produced less lactate thanthe Ctr and I1.5 groups (figure 3.1(b)). The differences were statistically significant inall cases (p < 0.05) except for the difference in lactate production between the Ctr andI1.5 group.

Table 3.1: The equations and correlation coefficients of the linearly fitted regression curves ofthe glucose consumption and lactate production of the Ctr, I12 and I1.5 groups.

Glucose R2 Lactate R2

Ctr y=0.1042x-0.8502 0.9829 y=0.1319x+3.9979 0.9282I12 y=0.0749x+0.1321 0.9799 y=0.0854x+5.2299 0.6509I1.5 y=0.1064x+0.9398 0.9658 y=0.1252x+6.2026 0.7342

The glucose consumption rates (nmoles (106 cells)−1 h−1) for the time period from t=16to t=71 hours were calculated by dividing the amounts of consumed glucose, which werecalculated using the regression curves, by the the initial number of cells per constructand the time span (Equation 3.1). The same equation was used for calculating thelactate production rates. The calculated rates and the ratios of the produced lactateover the consumed glucose are shown in table 3.2. For comparison, the relative ratesbased on the Ctr and I1.5 groups are also shown in table 3.2. The I12 group has aslower glucose consumption and a significantly slower lactate production than the Ctrgroup. The I1.5 group consumed glucose and produced lactate in a similar way as theCtr group. Comparing the two loading regimes, the I12 group had a significantly slowerglucose consumption and significantly slower lactate production than the I1.5 group.

Amountt=71 (µmol) - Amountt=16 (µmol)

9.955 · 106 cells · 55 hours·1015 = rate (nmoles (106 cells)−1 h−1) (3.1)

Table 3.2: The glucose consumption and lactate production rates (nmoles (106 cells)−1 h−1) ofthe Ctr, I12 and I1.5 groups. A comparison, using relative rates, is made betweenthe three groups. * Indicates significant difference (p < 0.05).

Glucose consumption Lactate production Ratio(nmoles(106 cells)−1h−1) (nmoles(106 cells)−1h−1) (lactate/glucose)

Ctr 10.47 13.25 1.27I12 7.52 8.58 1.14I1.5 10.69 12.58 1.18

Relative rates Relative ratesI12 vs Ctr 0.72 · Ctr 0.65 · Ctr∗

I1.5 vs Ctr 1.02 · Ctr 0.95 · CtrI12 vs I1.5 0.70 · I1.5∗ 0.68 · I1.5∗

3.2 Biochemical analysis

The average amounts (µg per dry weight) of GAG and DNA of control Ctr(t=0) samplesand the unloaded control (Ctr(t=138)) and intermittently loaded (I12(t=138), I1.5(t=138))

16

groups are shown in figure 3.2(a) and 3.2(b) respectively. The average amount of GAGin the I1.5(t=138) group is higher than in the other groups, although this increase isnot significant. Over the culture time the amount of DNA increased in all the groups.The I12(t=138) group showed the biggest increase, but only the increase of the Ctr(t=138)

group was significant (p<0.05). The GAG productions µg per µg DNA were calculatedby subtracting the initial (Ctr(t=0)) amount of GAG from the t=138 groups, and thendividing the differences by the amount of DNA (figure 3.3). It can be seen that the onlygroup that produced GAG is the I1.5(t=138) group.

0

2

4

6

8

10

12

µg G

AG

/ m

g dr

y w

eigh

t

Ctr(t=0) Ctr(t=138) I12(t=138) I1.5(t=138)

(a)

0

0.5

1

1.5

2

2.5

3

3.5

4

µg D

NA

/ m

g dr

y w

eigh

t

*

Ctr(t=0) Ctr(t=138) I12(t=138) I1.5(t=138)

(b)

Figure 3.2: Amount (µg per mg dry weight) of GAG (a) and DNA (b) for Ctr(t=0) samples(n=4), Ctr(t=138)(n=7), I12(t=138)(n=1) and I1.5(t=138)(n=2) groups. * Indicatessignificant difference (p < 0.05).

−0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

µg G

AG

pro

duct

ion

/ µg

DN

A

*

Ctr(t=138) I12(t=138) I1.5(t=138)

Figure 3.3: Amount of produced GAG (µg per µg DNA) of Ctr(t=138), I12(t=138) and I1.5(t=138)

groups. * Indicates significant difference (p < 0.05).

17

3.3 Histology

Staining with hematoxylin/eosin clearly stained the cells of the constructs. The sec-tions of the Ctr(t=0) constructs showed a homogenous cell distribution (figure 3.4). Theslides from the different groups (Ctr(t=138), I12(t=138), I1.5(t=138)) showed no differencesin matrix formation.

Figure 3.4: Tissue section on day 0, stained with hematoxilin/eosin.

18

Chapter 4

Discussion

This study demonstrates different rates of glucose consumption and lactate production ofchondrocytes subjected to different dynamic loading regimes, which are known to resultin differences in proliferation and ECM synthesis. Measuring the energy metabolism istherefore useful in tissue engineering of cartilage for monitoring chondrocyte activity.Tissue engineering of cartilage generally takes weeks, because chondrocytes have a slowmetabolism. For example, chondrocyte/agarose constructs need dynamical loading forat least 28 days before significant differences with control constructs can be found inGAG content and Young’s modulus.11 Within such long culture periods, measurementsbased on a three days time period, like in this study, can provide enough feedback foractive control, i.e. to adjust culture conditions. However, the metabolic rates are not yetcoupled quantitatively to the GAG and DNA contents of the constructs, meaning thatthere are no criteria when to change or stop the dynamical loading. Thus, the metabolismcan be used to monitor the chondrocytes activity, but more experiments should be done,before a control system based on the energy metabolism can be developed.

In this study the glucose consumption and lactate production rates were determined fromthree days culture periods, at the end of which almost all glucose from the 2 ml mediumwas consumed. Metabolic rates will be less accurate when the metabolic concentrationsin the medium change less rapidly.

The average glucose consumption and lactate production of all the groups, 9.56 and11.47 nmole (106 cells)−1 h−1 respectively, are 3-10 times lower than the rates found inthe literature.21,33,34 This can be explained by the fact that after cell isolation, the cellswere only equilibrated in medium for 1 hour before starting the experiment so it could bepossible that the cells were not adjusted to their new culture environment. In literature,constructs generally are equilibrated for 24 or 48 hours, before starting the experiments.Moreover, a post-hoc analysis of glucose concentrations in the construct after leaving itin medium for 1 hour shows that the glucose concentration in the construct is not yet inequilibrium (figure C.2).

The ratios of produced lactate over consumed glucose over the period of t=16 tillt=71 hours (1.27, 1.14 and 1.18 of the Ctr, I12 and I1.5 groups respectively), are lowcompared to the literature. Ratios of 1.65-2.34 are reported in short period (1-4.5 hours)measurements using cartilage explants.21,34 Similar ratios were reported using chondro-cytes seeded on polymer scaffolds after 4-5 weeks of culturing.33,36 When the ratios in

19

this study are calculated based on the period from t=0 till t=71 hours, thus taking astarting point of 0 mM consumed glucose and 0 mM produced lactate, the ratios become2.04, 2.07 and 1.78 for the Ctr, I12 and I1.5 groups respectively. Although these ratiosare comparable with the literature, they are incorrect, because the ratios are clearlyinfluenced by the high lactate production in the first 16 hours. Hence, caution must betaken when interpreting ratios based on single time point measurements.

For the first period of 16 hours, more lactate was produced than could be accountedfor by the depletion of glucose (ratios are 11.58, 5.32 and 3.18 for the Ctr, I12 and I1.5groups respectively). Lee et al.21 also found high ratios (3.96±2.34) when culturingbovine cartilage explants under anaerobic conditions. In the present study it is unlikelythat the high ratios are caused by an anaerobic environment, because all groups have ahigh lactate production, including the Ctr group which was cultured in 12 wells plateswhich have a good oxygenation. An explanation could be that the chondrocytes are stilladjusting from the transfer of their natural environment to the construct, which causesthe consumption of internal carbon sources, like glycogen,43 instead of external glucose.

It was hypothesized that the glucose molecules, used for GAG synthesis, would notyield lactate molecules and this would change the ratio. It is obvious that when glucose isdegraded through aerobic glycolysis there is also no lactate production. To discriminatebetween GAG synthesis and aerobic glycolysis, the O2 consumption of the chondrocytescan be measured. Appendix A discusses the use of microelectrodes for measurement ofadditional discriminating metabolites.

Surprisingly, the initial amount of glucose in the medium was significantly differentbetween the first and second experiment, 10.06 µmol (5.03 mM) and 8.50 µmol (4.25mM) respectively. The amount of glucose in two additional aliquots of medium weremeasured, which resulted in a mean of 8.85±1.06 µmol (4.43±0.38 mM). It is unlikelythat the large standard deviation is caused by the glucose assay. How these differenceswere introduced in the medium is however unclear. In following experiments, it is betterto use bathes of medium with the same glucose concentration to ensure equal startingpoints. The glucose consumption rate of chondrocytes is non-linearly dependent on theglucose concentration, but the non-linearity is only visible at very low glucose concen-trations (0-0.7 mM).34,47 Hence, although the effects of the glucose concentration on theconsumption rates will not influence the rates in the first three days of the experiment,the cells from the second experiment will reach a critical glucose concentration earlier,which then influences the metabolic rates.

The correlation coefficients of the lactate regression curves are lower than those of theglucose curves. This is mainly caused by the surprising finding that the averages of pro-duced lactate of the three groups at t=47 hours are lower than at t=40 hours. The sameis observed at t=71 and t=64 hours. Although lactate consumption has been reported,when lactate was used as sole substrate for cartilage explants and the concentration washigher than 10mM,34 it is unlikely that the cells in the present study consumed lactate.If utilization of lactate occurred, this would also be expected in the period from t=71till t=138 hours, but in this period the amount of lactate still increases for the Ctr andI1.5 groups.

The facts that the I1.5 group had the highest GAG production and that the I12 grouphad the highest amount of DNA, indicate that the different intermittent loading regimesdid direct the cells to two different processes, although both increases are not significant.This insignificance is caused by the small number of data points per experimental con-

20

dition, which is caused by the low number of available bioreactors. Surprisingly though,our results are opposite to the findings of Chowdhurry et al.,4 who reported the highestPG synthesis in the I12 group and the highest proliferation in the I1.5 group. Possi-bly, differences between the protocols of the experiments and the loading devices areresponsible. Chowdhurry et al.4 used bovine chondrocytes/agarose constructs with acell density of 4·106 cells ml−1, which were equilibrated in buffered medium for 24 hoursand then loaded for 48 hours. In the present study the same medium was used for 5.5days without pH buffers, to enable future pH measurements. It may well be that the pHlowered during the culture period and that this slowed the metabolic processes.

The GAG production of the I1.5 group was 0.97±0.71 µg GAG per µg DNA after 5.5days of culture. Lee et al.20 reported values of 2-3 µg GAG per µg DNA after 4 days ofculture, but the amount of released GAG in the medium was included in these values,so the values can not be compared. When the GAG content is calculated in percentwet weight (% ww), the value for the I1.5 group is 0.046±0.015% ww. Mauck et al.25

reported GAG values of chondrocyte/agarose constructs (20·106 cells ml−1 in 2% (w/v)agarose) of 0.51% ww after 7 days of culture. This difference is too big to be explained bythe 2 day difference in culture time. The lower amount of GAG could again be explainedby the fact that the cells were only equilibrated for 1 hour compared to 48 hours.25

Besides dynamical loading of the chondrocytes, the movement of the magnet also causesmixing of the medium in the bioreactor. To see what kind of influence this mixing hason the metabolite concentrations in the chondrocyte/agarose constructs a computationalanalysis is performed (Appendix C). A 1D finite element mesh is used in which the glu-cose consumption of the cells and the glucose diffusion in the medium and construct aremodelled. Mixing of the medium is modelled by a 10-fold increase of the glucose diffu-sion rate in the medium and is switched on and off in the same sequence as the loadingregimes. The equilibration hour and the first 71 hours of the experiment were simulated,but without removing medium during this period. The results show (Appendix C) thatmixing alone causes slight differences in glucose concentrations in the construct betweenthe I12 and I1.5 groups, but these differences are not in the non-linear areas of the glu-cose consumption. Hence, it can therefore be concluded that the changes in metabolismfound in this study are not influenced by the mixing of the medium.

In summary, this study demonstrates different rates of glucose consumption and lactateproduction of chondrocytes subjected to different dynamic loading regimes. The I12group consumed glucose and produced lactate at a lower rate than the Ctr and I1.5groups. Furthermore it is demonstrated that caution must be taken when interpretingratios of produced lactate over consumed glucose over short periods of time. It is shownthat the ratios calculated over the time period of t=16 till t=71 hours are much lowerthan the ratios calculated over the t=0 till t=71 hours period. When taking these timedependent effects into account, ratios are different between the Ctr, and the I12 andthe I1.5 groups. In addition, differences in DNA content and GAG production betweenthe I12 and I1.5 groups are found. Of major importance in the latter issue is the factthat apparently similar stimulation protocols between the latter study and the study ofChowdhurry et al.4 show opposite effects in proliferation and ECM synthesis.

The combined results indicate that the energy metabolism is different when the cellsare either proliferating or synthesizing ECM. Measuring chondrocyte metabolism is con-cluded to be a promising tool to monitor cartilage TE, although monitoring metabolismalone is insufficient for a control system.

21

Chapter 5

Future research andrecommendations

In this study, differences in the glucose consumption and lactate production of chon-drocytes were found between two dynamical loading regimes. These differences candistinguish the chondrocytes when they are proliferating or synthesizing ECM, but ac-tive control of the tissue engineering process is not yet possible by measuring the energymetabolism alone. It is therefore recommended to investigate this metabolism in moredetail. Measuring the O2 and glutamine consumption and the NH3 production wouldgive more discrimination between GAG synthesis and aerobic glycolysis. This GAG syn-thesis can be determined more accurately, when the GAG content of the medium is alsomeasured. Additional pH measurements can be used to monitor the state of the medium.It was hypothesized that some differences in metabolic rates and construct propertieswith the literature were caused by the fact that the cells were not equilibrated in mediumfor 24 hours. For future research, the protocol should thus be adjusted by expanding theequilibration period.

Although different lactate over glucose ratios are found between the different groups,they were very low compared to the literature. Due to the low value they cannot belinked to differences in GAG production directly. It is recommended to analyse thoseratios after the longer equilibration period and over longer culture periods. The values ofthe ratios will then probably be more comparable with literature and better conclusionscan be drawn. Furthermore, the glucose concentration in the bioreactor should be kepthigher during a dynamical loading experiment, to ensure a more constant chondrocytemetabolism. This can be done by replacing the medium samples by fresh medium.

22

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[35] Pullens R.A.A. Cartilage tissue engineering in a bioreactor. A literature review.Technical Report BMTE03.33, TU/e, 2003.

[36] Saini S. and Wick T.M. Concentric cylinder bioreactor for production of tissue en-gineered cartilage: Effect of seeding density and hydrodynamic loading on constructdevelopment. Biotechnology Progress, 19:510–521, 2003.

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26

Appendix A

Microelectrodes

If it is desirable to measure more parameters of the energy metabolism, like pH andO2, CO2 and NH3 concentrations, microelectrodes can be used. For bioreactors flow-thru electrodes (figure A.1) are very convenient, because they can be connected to thetubing of the bioreactor system. For the IMBIOTOR system electrodes were selected,which fit tubing with an inner diameter of 1/16 inch (1.58 mm). An example of a totalsetup using the IMBIOTOR combined with flow-thru electrodes and a pump is shownin figure A.2. In this appendix the use of the electrodes for the measurement of pHand O2 concentration is discussed. The electrochemical working of the electrodes willbe explained as well as the calibration procedure. Furthermore the implementation withthe bioreactor system will be discussed.

Figure A.1: Example flow-thru electrode

Z

N

RESERVOIR

� � �� � �� � �

� � �� � �� � �

O2

A/D board −− Computer

CO2ReferenceBIOREACTOR

Z N

Pump

NEW MEDIUM

MV−ADPT

pH NH3

O2−ADPTMV−ADPTMV−ADPT

Figure A.2: A schematic overview of a bioreactor system with flow-thru microelectrodes.

27

A.1 pH measurement

For measuring pH, a pH and a reference electrode are used. This measurement is basedon the basic phenomenon of the development of an electrical potential (voltage) by achemical reaction in both electrodes. The pH measuring electrode is a hydrogen ionsensitive glass bulb, with a millivolt output that varies with the changes in the relativehydrogen ion concentration inside and outside of the bulb. The reference electrode isa concentration element, an electrochemical millivolt generator, that as such providesthe amplifier of the system with the reference potential necessary. This electrode ideallymaintains a constant potential, regardless of other species in solution. It must alsoestablish electrical connection to the measurement electrode through the water to closethe circuit, because without this connection the measurement cannot be made. Stabilityand non-selectivity are maintained by making electrical contact between the pH andreference electrode via an inert salt bridge. Typically the salt bridge is composed ofconcentrated potassium chloride, the same salt used to form the Ag/AgCl electrode.This electrical contact must allow uninhibited movement of electrolyte between the pHand the reference electrode to assure a repeatable constant reference potential. At thesame time it must not grossly contaminate the sample with electrolyte. Therefore, arestriction (a porous ceramic frit) is used to slow the flow. The internal resistance ofthe pH electrode is very high, which produces only a very small current with which tomeasure the voltage. This means that a high impedance amplifier has to be used tobe able to measure the voltage change. After the amplification the voltage differenceis recorded by acquisition hardware in a computer. A labview program was writtento record the signals and calculate the pH. The pH measurement is very sensitive toelectrical noise disturbance, which is caused by the high impedance of the glass andreference electrode circuit, which permits the easy induction of electrical signals causedby the electro-magnetic fields generated by other electrical equipment in the vicinity.The signal from the amplifier had to be filtered with a low pass RC filter (time constant= 2.5 minutes) to suppress 50 Hz noise from the low voltage net.

pH calibrationThe pH electrodes were calibrated using three buffer solutions (pH 4, 7 and 9). Eachbuffer solution was added to the bioreactor system with the electrodes connected. Thebuffer solution was then pumped round and an average value was calculated from 10seconds of voltages readings (sampled with 500 scans per second). This was done every10 minutes for 1.5 hours. The calibration curve is shown in (figure A.3).

3 4 5 6 7 8 9 10−2

−1

0

1

2

3

4

pH

Vol

tage

(V

)

Figure A.3: Calibration curve pH

28

ImplementationThe pH electrodes were installed in the medium tube between the bioreactor and thepump. The pH was measured for 20 seconds within the incubator. One problem whicharose was the fact that the signal was influenced by the roller pump. The sinusoidalflow pattern could be seen in the voltage signal of the pH electrodes (figure A.4). Thisproblem was solved by implementing a routine in the labview program to stop the pumpjust before acquiring the readings of the pH electrodes.

0 5 10 15−2

−1

0

1

2

3

4

Time (sec)

Vot

age

(V)

(a) Pumplevel 1

0 5 10 15−2

−1

0

1

2

3

4

Time (sec)

Vot

age

(V)

(b) Pumplevel 5

0 5 10 15−2

−1

0

1

2

3

4

Time (sec)

Vot

age

(V)

(c) Pumplevel 9

0 5 10 15−2

−1

0

1

2

3

4

Time (sec)

Vot

age

(V)

(d) Pump switched off

Figure A.4: Voltage signals of the pH electrodes with the roller pump running at levels 1, 5 and9 (a, b and c) and when the pump was switched off (d).

A second issue is the temperature sensitivity of the pH electrodes. When the voltage ofa pH 4.01 buffer was recorded for ±1 day inside (T=37◦C) and outside (T=18◦C) theincubator (figure A.5) a ± 0.5V difference, (pH of ± 0.7), can be found.

Even though all these small problems were solved, the first attempts to monitor the pHin the bioreactor during culturing of chondrocyte/agarose constructs were unsuccessful.The constructs were made to a final cell concentration 5·106 cells ml−1 in 3% (w/v)agarose and cultured for 138 hours. The pH stayed fairly constant in the first days(figure A.6), but at the end the pH seemed to rise, which is very unlikely physiologically,because the medium was not changed for the entire culture period, which means thatthe pH should be much lower due to waste products of the chondrocytes. During thecultured period the pH was also monitored using a paper pH indicator, which indicateda lowered pH of 6.4 at t=138. When the electrodes were afterwards used to measure the

29

0 0.5 1 1.51

1.5

2

2.5

3

3.5

Time (days)

Vol

tage

(V

)

Outside

Inside

Figure A.5: Temperature sensitivity of the pH electrodes. Voltage signal from the pH elec-trodes, measured inside (T=37◦C) and outside (T=18◦C) the incubator.

pH’s of the buffer solutions they still gave the same respons, meaning that the membraneor ceramic plug was not blocked by proteins or other substances in the medium, whichcould cause the drift in the signal.

0 1 2 3 4 55.5

6

6.5

7

7.5

Time (days)

pH (

−)

Period 1Period 2Period 3Period 4

Figure A.6: Measurement of pH during culture of chondrocyte/agarose construct.

Using the electrodes in this particular study was also difficult, because a very low amountof medium could be used in the bioreactor. When the electrodes and pump were con-nected to the bioreactor a certain amount of volume went into the tubing, the lowamount of medium was not sufficient to keep the construct wet. After the measurementit was difficult to get all the medium out of the tubes and back into the bioreactor. Theprocedure of connecting, measuring and sterilely reconnecting the tubes and electrodesapproximately takes one hour. Repeating the procedure for four bioreactors resulted inpH measurements which could not be compared, because of the long time span betweenthe measurements.

A.2 O2 measurement

The O2 sensor is a clarke type polaragraphic sensor which uses a platinum cathodesealed in glass that is held at a polarization voltage of -0.8 V. O2 diffuses through themembrane in at the cathode it is electrochemically reduced to hydroxide. The anodeis a silver tube. The greater the O2 partial pressure, the more O2 diffuses through themembrane in a given time. This results in a current that is proportional to the O2 inthe sample. An O2 adapter is used to convert the very small current (picoAmpere) tomillivolt. For measuring O2 consumption it is obvious that the system has to be sealed

30

of from the environment so that it is not possible for O2 to diffuse into the system. Inorder to achieve this, the O2 electrode is located closest to the outflow of the bioreactorand connected to the bioreactor via a special tubing (Pharmed) which has a much lowerpermeability for O2, CO2 and N2 than normal silicone tubing.

O2 calibrationThe O2 electrode was calibrated using a glas bottle with H2O which was bubbled for atleast 30 minutes with air or N2 to obtain an O2 concentration of 21% and 0% respectively.These solutions were then pumped through the electrodes and an average value wascalculated from 4 seconds of voltages readings (sampled with 500 scans per second).This was done every 5 minutes for 1 hours. Two calibration points (figure A.7) werederived from these measurements.

0 21−0.35

−0.3

−0.25

−0.2

−0.15

−0.1

−0.05

0

O2 concentration (%)

Vol

tage

(V

)

Figure A.7: Calibration points O2 electrode.

31

Appendix B

Protocols

B.1 Materials

Table B.1: ChemicalsChemical Catalogue number and supplier1,9 Dimethyl-Methylene Blue 34,108-8 Sigma-Aldrich CompanyLow gelling Agarose type VII A9045, Sigma-Aldrich CompanyChondroitin Sulfate from bovine trachea 27042, FlukaCollagenase type I C0130, Sigma-Aldrich CompanyDublecco’s Modified Eagle’s Medium D2902, Sigma-Aldrich CompanyD(+)-Glucose 1.08337.1000, MerckEDTA disodium salt ED2SS, Sigma-Aldrich CompanyFetal Bovine Serum (Heat inactivated (Appendix B.4)) S0113, Biochrom AGFormic acid 06440, FlukaGlucose Oxidase/Peroxidase Reagent G3660, Sigma-Aldrich CompanyL-(+)-Lactic Acid L1875, Sigma-Aldrich CompanyL-cystein hydrochlorid C1276, Sigma-Aldrich CompanyLactic Acid Reagent TB-735-10, Kordia Life Scienceso-Dianisidine Reagent D2679, Sigma-Aldrich CompanyPapain P5306, Sigma-Aldrich CompanyPenicillin / Streptomycin A2213, Biochrom AGPhosphate Buffered Saline (PBS) P4417, Sigma-Aldrich CompanySigma GenElute Mammalian Genomic DNA Kit G1N70, Sigma-Aldrich CompanySodium Acetate Trihydrate S7670, Sigma-Aldrich Company

The culture medium has the following composition:

Contents Percentage v/vDMEM 89 %(dulbecco’s modified eagle’s medium, 1 g/ml glucose,

wo sodium bicarbonate and phenol red, Sigma)

Fetal Bovine Serum 10 %(Biochrom)

Penicillin/Streptomycin 1 %(10000 µg/ml, Biochrom)

32

B.2 Cell harvesting protocol

Materials:

• 70% ethanol

• sterile scalpels and blades

• sterile PBS

• medium

• collagenase

• falcon tube

• cell dissociation sieve (Sigma)

• cell counting tools

Protocol:

• Remove excessive dirt by brushing the paw under tap water.

• Sterilize paw in extran soap solution for half an hour.

• Prepare a collagenase solution by dissolving 1 mg collagenase type I (C0130, Sigma)per ml DMEM.

• Make sure that the next steps are performed sterilely in a LAF cabinet.

• Remove the skin over the joint the cartilage is to be harvested from, by making anincision in the skin below the joint and stripping of the skin.

• Open the joint and follow the joint surface with the scalpel and cut all the tendons.

• Take away the cartilage and put it into the tube with the collagenase solution.

• Attach the falcon tube to a shaker and put it in an incubator at 37 ◦C overnight.

• Use a cell dissociation sieve to remove undigested tissue.

• Add the solution with the cells to a falcon tube.

• Centrifuge for 7.5 minutes at 2000 rpm and remove the supernatant.

• Wash the cells by adding 20 ml PBS and centrifuge for 7.5 minutes at 2000 rpmand remove the supernatant.

• Repeat the previous step.

• Add 1 ml of medium, resuspend the cells and count the cells.

B.3 Obtaining an agarose gel seeded with chondrocytes

Materials:

• low gelling temperature agarose, type VII (A9045, Sigma)

• rock ’n’ roller in oven at 45oC

• PBS

• sterile scalpel blades

• sterile glass tube with screw lit

• warm pipets

• sterile moulds and bioreactors (sterilization with alcohol overnight)

• warm water bath (37-40◦C) in the LAF hood with a tube holder

33

Protocol:

• Prepare the cell suspension (50·106 cells ml−1).

• Put the cell suspension in the incubator during the preparation of the agarosesolution, so the cell suspension has the right temperature.

• Make a 6% (w/v) agarose solution (6 gram agarose per 100 ml PBS).

• Autoclave the agarose solution in a tube of glass (liquid cycle).

• Put the tube with agarose on a roller in the oven at 45oC.

• Put the moulds in the LAF cabinet.

• Put the well mixed cell suspension in the LAF cabinet.

• Add the cell suspension to the agarose solution, mix thoroughly.

• Fill the moulds with the agarose/cell mixture by using a warmed pipet.

• Put the moulds for 20 minutes in the fridge.

• Remove the samples from the moulds with a scalpel and put the samples in apetridish or in the bioreactor and place them in the incubator.

B.4 Protocol heat inactivation of fetal bovine serum

• Defrost a bottle of FBS (S0113, Biochrom AG) till room temperature.

• Fill one empty FBS bottle with 100 ml H2O.

• Place the thermometer in the bottle with H2O.

• Place both bottles in a water bath.

• Heat the solutions till 56 ± 0.5 ◦C.

• Note: during heating shake bottle every 5 mins.

• Keep temperature constant for 30 mins.

• After 30 mins immediately cool bottles in ice.

B.5 Glucose assay protocol

The glucose assay protocol is based on the protocol from the glucose assay kit (GAGO-20) from Sigma. From this kit the Glucose Oxidase/Peroxidase Reagent (G3660) ando-Dianisidine Reagent (D2679) are used.

Materials:

• Standard: Make a stock solution of 5.55 mM glucose by adding 25 mg D(+)-Glucose (1.08337.1000, Merck) ) to 25 ml H2O.

• Glucose assay: Add 0.8 ml of the o-Dianisidine Reagent to the amber bottlecontaining the 39.2 ml of Glucose Oxidase/Peroxidase Reagent. Solution is stableup to 1 month at 2-8 C.

• Prepare the standards from the stock solution:

34

Stock H2O Concentration0 ml 5 ml 0 mM1 ml 4 ml 1.11 mM2 ml 3 ml 2.22 mM3 ml 2 ml 3.33 mM4 ml 1 ml 4.44 mM5 ml 0 ml 5.55 mM

Protocol:

• Dilute the samples and standards 1:10 (add 20 µl sample to the wells of a wellsplate and add 180 µl H2O)

• Mix the wells thoroughly.

• Pipet 20 µl of the diluted samples into the wells of a 96 wells plate.

• At zero time, start the reaction by adding 40 µl of assay reagent to the first row ofwells using a multi channel pipet. Allow a 10 second interval between additions ofassay reagent to each subsequent row.

• Put the plate in the incubator for exactly 30 minutes at 37 ◦C.

• Stop the reaction at 10 second intervals by adding 40 µl of 12 N H2SO4.

• Mix the wells by placing the plate on a shaker.

• Measure the absorbance against the reagent blank at 540 nm.

• Determine the standard curve and calculate the glucose concentrations.

0 0.1 0.2 0.3 0.4 0.5 0.6

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Glucose concentration (mM)

Inte

nsity

(−

)

y = 0.0586xR2 = 0.9872

Figure B.1: Calibration curve of glucose assay.

B.6 Lactate assay protocol

Materials:

• Stock solution: Add 84.265 µl L-(+)-Lactic Acid Solution (L1875, Sigma) to 25ml H2O to get a stock solution (12 mM). Note: Lactic Acid Solution is 30% aqueous.

Calculation:

25 ml H2O · 12 mmol/l · 90.08 mg/mmol = 27.024 mg lactic acid

27.024 mg

1.069 mg/µl·100

30= 84.265 µl lactic acid solution

35

• Prepare the standards from the stock solution:

Stock H2O Concentration1 ml 0 ml 12 mM2 ml 1 ml 8 mM1 ml 1 ml 6 mM1 ml 2 ml 4 mM1 ml 5 ml 2 mM0 ml 1 ml 0 mM

• Lactate Reagent: Prepare lactate reagent (TB-735-10, Kordia Life Sciences) byadding 10 ml purified H2O to one bottle.

Protocol:

• Dilute the samples and standards 1:10 (add 180 µl H2O to 20 µl sample).

• Pipet 10 µl diluted sample into the wells of a 96 wells plate.

• Pipet 100 µl lactate reagent into the wells.

• Measure the absorbance at 540 nm after 10 minutes.

• Determine the standard curve and calculate the lactate concentrations.

0 2 4 6 8 10 12 14

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Lactate concentration (mM)

Inte

nsity

(−

)

y = 0.0287xR2 = 0.9857

Figure B.2: Calibration curves of lactate assay.

B.7 GAG assay

Materials:

• Standard: Chondroitin sulfate (bovine trachea, Fluka 27042) stock solution (10mg/ml in aliquots of 100 µl stored in -20 ◦C).

• 0.5 M EDTA Stock solution: 1.8 gram ethylenediaminetetraaceticacid disodiumsalt dihydrate (ED2SS, Sigma). Add MQ up to a volume of 10 ml. EDTA dissolvesat a pH of 8, so first titrate the pH at 8 with NaOH.

• Unfinished papain solution: Add 200 ml MQ to 68 gram sodium acetate tri-hydrate (S7670, Sigma) and 0.79 gram L-cystein hydrochlorid (C1276, Sigma).Titrate the pH at 5.6 with HCl. Add 5 ml of 0.5 EDTA stock solution and fill upto 250 ml with MQ.

36

• DMB solution: Dissolve 1.6 mg DMB (1,9 Dimethyl-Methylene Blue, 34,108-8,Sigma) in 0.5 ml ethanol. Add 0.2 ml 2M sodium acetate (dissolve 27.2 gr sodiumacetate in 100 ml MQ for 2 M solution) and 200 µl formic acid (06440, Fluka).Add 80 ml MQ and titrate the pH at 3.5. This solution has to be stored at 4 ◦Cprotected against light and will be stable up to 6 months.

Protocol:

• Determine wet weight of the samples.

• Lyophilize the tissue (overnight) and determine dry weight. Make sure you havepieces of at least 2 mg.

• Dilute the chondroitin sulfate 1:10 (add 900 µl to the 100 µl aliquot) up to 1 mg/ml.

• Prepare the standards in duplo:

– 30 µg: 30 µl standard solution (1 µg/µl)– 25 µg: 25 µl standard solution (1 µg/µl)– 12 µg: 12 µl standard solution (1 µg/µl)– 6 µg: 6 µl standard solution (1 µg/µl)– 4 µg: 4 µl standard solution (1 µg/µl)– 2 µg: 2 µl standard solution (1 µg/µl)– 0 µg: nothing

• Take 25 ml of the papain solution (or as much as you need) and add 3.5 mg papain(P5306, Sigma, stored in -20 ◦C).

• Add 1 ml papain solution to the standards and your samples and shake gently.

• Incubate the standards and the samples overnight at 60 ◦C (about 16 hours).

• Stop the digestion by heating up to 95 ◦C for one hour.

• Vortex the samples and centrifuge at 13000 rpm for 8 minutes.

• Pipet 40 µl per sample into the wells of a 96 wells plate in duplo.

• Add 150 µl DMB solution to each well, mix well on a shaker shortly and measureimmediately within 10 minutes.

• Determine absorbance at 540 nm and 595 nm and extract these values (540-595).

• Determine the standard curves and calculate the GAG concentrations.

−5 0 5 10 15 20 25 30 35−0.4

−0.35

−0.3

−0.25

−0.2

−0.15

−0.1

−0.05

0

0.05

0.1

GAG concentration (µgram/ml)

Abs

orba

nce

diffe

renc

e (5

40−

595n

m)

y = 0.0126x − 0.3278R2 = 0.9979

Figure B.3: Calibration curve GAG assay.

37

B.8 DNA assay

Mammalian cells and tissues are lysed with a chaotropic salt containing buffer to ensuredenaturation of macromolecules. DNA is bound to the spin column silica-based mem-brane and the remaining lysate is removed by centrifugation. A filtration column is usedto remove cell debris, after washing to remove contaminants; the DNA is eluted withbuffer into a collection tube.

Materials:

• Sigma GenElute Mammalian Genomic DNA Kit (G1N70)

• 96% Ethanol

• TE buffer: 10 mM Tris-HCl, 1mM EDTA, pH 8-8.5:Dissolve 121 mg Tris and 37 mg EDTA in 80 ml MQ. Titrate the pH at 8-8.5 usingHCl and fill up to 100 ml with MQ.

• Harrier Centrifuge equipped with a capped rotor for 1.5 ml tubes.

Protocol:

• Determine wet weight of the samples.

• Lyophilize the tissue and determine dry weight. Make sure you have pieces of atleast 2 mg.

• Set the heating block at 55 ◦C.

• Transfer the tissue to the special assay eppendorf cups (with the orange lid).

• Add 180 µl Lysis Solution for Tissue (B6678) and add 20 µl proteinase K solutionto the samples.

• Vortex and incubate (shaking) at 55 ◦C for at least 4 hours (check whether thedigestion is complete).

• Set the heating block at 70 ◦C.

• Add 200 µl Lysis Solution (B8803) to the samples and vortex 15 sec.

• Incubate at 70 ◦C for 10 minutes.

• Add 200 µl 96% ethanol to the samples and vortex 5-10 sec.

• Assemble the binding columns (red o-ring) with the 2 ml collection tubes.

• Transfer 500 µll of the contents of the tubes into the binding columns (the totalvolume in the tube is 600 µl, but you will correct for this later).

• Centrifuge at >6500 g for 1 minute.

• Replace the collection tube.

• Add 500 µl 1X Wash Solution to the columns.

• Centrifuge at >6500 g for 1 minute.

• Replace the collection tube.

• Add 500 µl 1X Wash Solution to the columns.

• Centrifuge at 12000-16000 g for 3 minutes.

• Replace the collection tube.

38

• Pipet 200 µl Elution Solution directly into the center of the column.

• Incubate for 5 minutes at room temperature.

• Centrifuge at >6500 g for 1 minute.

• Determine the absorbance at 260 and 280 nm using the quartz cuvettes. Rinse thecuvettes with TE buffer before use and re-use. Do not forget to measure the blanco(elution solution).

• Substract the blanco from all values and determine the ratio 260:280 nm, thisshould be 1.7-1.9, because this is the ratio when measuring pure DNA.

• An absorbance of 1.0 at 260 nm corresponds to 50 µg/ml double-stranded DNA.Determine the amount of DNA in the samples:

(Extension value at 260 nm * 10) / weight of the sample in mg

You have to correct for the fact that you only took 500 µl instead of 600 µl of thesample (step 11), so divide the amount of DNA by 500 and multiply by 600.

39

Appendix C

Computational analysis

In the present study, the chondrocyte/agarose constructs were placed in 2 ml of mediumfor 1 hour, in order to equilibrate the glucose concentration in the construct. The questionis whether the glucose concentration has indeed reached an equilibrium in that hour.

The diffusion of glucose can be estimated with the characteristic diffusion time (τ).This estimate is calculated by dividing the square of the diffusion distance (h), height ofthe construct, by the diffusion coefficient (D) of glucose through agarose,10,23 equationC.1. The value of τ is larger than 1 hour meaning that the glucose concentration hasnot reached an equilibrium.

τ =h2

D=

(3·10−3m)2

(2.88·10−6m2h−1)= 3.125 hours (C.1)

In the calculation of τ the glucose consumption by the cells is not taken into account.In order to investigate the effect of the glucose consumption on the equilibrium a com-putational analysis was performed. A 1D finite element mesh containing medium (15.07mm) and the chondrocyte/agarose construct (3 mm) was used (figure C.1).

15.07 mm 3 mm

b ca

Figure C.1: 1D finite element mesh. The top of the medium (a), the top of the construct (b)and the bottom of the construct (c) are indicated.

The glucose consumption within the construct is modelled with a fixed value based on theconstructs initial cell concentration (25·106 cells ml−1) multiplied by the experimentallydetermined glucose consumption rate (10 nmoles (106 cells)−1 h−1). Furthermore theglucose diffusion rates in agarose and in medium are implemented.23 Figure C.2 showsthe glucose concentration profiles in the medium and in the construct for 3.125 hours.

40

It can be seen that the glucose concentration in the construct has not reached a steadystate, due to the glucose consumption of the cells.

0 5 10 150

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Position (mm)

Glu

cose

con

cent

ratio

n (m

M)

a b c

Tim

e

Figure C.2: Glucose concentration profiles in the medium layer and in the construct for a periodof 3 hours.

Besides dynamically loading of the constructs, the movement of the magnet also causesmixing of the medium in the bioreactor. To see what kind of influence this mixing hason the metabolite concentrations in the construct, the first hour of equilibrating and thefollowing 71 hours of the dynamical loading experiment were simulated. At t=1 hoursthe glucose concentration of the medium is set back to its initial value, simulating thetransfer of the constructs to the bioreactors. Mixing of the medium is modelled by a10-fold increase of the glucose diffusion rate in the medium and is switched on and offin the same sequence as the loading regime. The glucose concentration profiles in themedium layer and the construct for I12 and I1.5 are shown in figures C.3(a) and C.3(b)respectively.

0 5 10 150

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Position [mm]

Glu

cose

con

cent

ratio

n [µ

M]

a b c

(a)

0 5 10 150

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Position [mm]

Glu

cose

con

cent

ratio

n [µ

M]

a b c

(b)

Figure C.3: Glucose concentration profiles in the medium layer and the construct for I12 (a) andI1.5 (b). The solid lines are from the equilibration hour (t=0 till t=1 hours) andthe dashed lines are from the dynamic loading experiment (t=1 till t=72 hours).

41

It can be seen that the profiles are different but that the mean values for the medium layerand the construct are more or less the same. Figure C.4 shows the glucose concentrationat the positions a, b and c in time. It can be seen that there are no large differences in thetime profiles. In the experiment performed in the present study the differences in glucoseconcentrations within the construct would have been even less, because glucose can alsodiffuse into the construct from the sides. Hence, it can be concluded that the changes inmetabolism found in this study are not influenced by the mixing of the medium.

0 10 20 30 40 50 60 700

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Time (hours)

Glu

cose

con

cent

ratio

n (m

M)

I12I1.5

(a)

0 10 20 30 40 50 60 700

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Time (hours)

Glu

cose

con

cent

ratio

n (m

M)

I12I1.5

(b)

0 10 20 30 40 50 60 700

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Time (hours)

Glu

cose

con

cent

ratio

n (m

M)

I12I1.5

(c)

Figure C.4: The glucose concentrations in time at the top of the medium (a), top of the con-struct (b) and bottom of the construct (c).

42