5B3FBd01 PDF

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Study of the influence of

pH on viability and
metabolism of chondrocytes
Rolf Pullens
BMTE 03.21
Supervisors:
Dr. J.P.G. Urban

Dr. C.C. van Donkelaar
Study of the influence of pH on viability and metabolism of chondrocytes
Contents
ABSTRACT ............................................................................................................................. 3
CHAPTER 1: INTRODUCTION .......................................................................................... 4

1.1 ARTICULAR CARTILAGE ................................................................................................... 4
1.2 METABOLISM AND GRADIENTS IN CARTILAGE ................................................................ 4
1.3 CULTURE SYSTEMS .......................................................................................................... 4
1.4 AIM AND OUTLINE............................................................................................................ 5
CHAPTER 2: MATERIALS AND METHODS ................................................................... 6
2.1 MATERIALS ...................................................................................................................... 6
2.2 LACTIC ACID .................................................................................................................... 6
2.2.1 Effect of lactic acid on pH in cartilage ..................................................................... 6
2.2.2 Influence of PEG on pH ............................................................................................ 7
2.3 CHONDROCYTES .............................................................................................................. 7
2.3.1 Tissue dissection........................................................................................................ 7
2.3.2 Tissue digestion......................................................................................................... 7
2.3.3 Cell counting ............................................................................................................. 8
2.3.4 Alginate beads........................................................................................................... 8
2.3.5 Releasing cells from beads........................................................................................ 8
2.4 VIABILITY STUDIES .......................................................................................................... 8
2.4.1 Viability experiment 1 ............................................................................................... 8
2.4.4 Lactate assay............................................................................................................. 9
2.5 PH GRADIENT IN DIFFUSION CHAMBER ............................................................................ 9
2.5.1 Experimental set-up .................................................................................................. 9
2.5.2 Carboxy SNARF-1 pH indicator ............................................................................. 10
2.5.3 Determination of the right concentration of dye ..................................................... 10
2.5.4 Calibration curve .................................................................................................... 11
2.5.5 pH gradient experiments ......................................................................................... 11
2.5.6 pH gradient around one cell ................................................................................... 12
2.6 NUMERICAL MODEL LACTIC ACID PRODUCTION ............................................................ 12
CHAPTER 3: RESULTS ...................................................................................................... 14
3.1 LACTIC ACID .................................................................................................................. 14
3.1.1 Effect of lactic acid on pH in cartilage ................................................................... 14
3.1.2 Influence of PEG on pH .......................................................................................... 15
3.2 VIABILITY STUDIES ........................................................................................................ 15
3.2.1 Viability experiment 1 ............................................................................................. 15
3.3 PH GRADIENT IN SANDWICH .......................................................................................... 16
3.3.1 pH gradient experiments ......................................................................................... 17
3.3.2 pH gradient around one cell ................................................................................... 18
3.4 NUMERICAL MODEL LACTIC ACID PRODUCTION ............................................................ 19
DISCUSSION......................................................................................................................... 20
REFERENCES ...................................................................................................................... 22
2
Department of Biomedical Engineering
Abstract
Articular cartilage is avascular and therefore nutrients have to diffuse into the cartilage to
the chondrocytes. Due to this diffusion limitation the cells in the deep zone of cartilage are
under low O2 and glucose concentrations and low pH compared to the surface cells. The main
goal of the research project was to investigate the influence of pH on the viability and
metabolism of the chondrocytes, on top of the influence of different conditions of low glucose
and low O2. For this purpose, articular chondrocytes were cultured under these different
conditions for three days and assayed for viability.
It was found that the viability of chondrocytes cultured without glucose started to die
immediately. Chondrocytes cultured at pH=6.2 had a lower viability than cells cultured at
pH=7.4. Culturing chondrocytes at low O2 concentrations did not have an effect on the
viability when glucose was present. In the absence of glucose the effect of low O2 was not
clear.
Diffusion chambers can be used for studying metabolic processes of cells, but it is
difficult to measure the pH in those chambers. Therefore the second main goal of this project
was to set up a method to measure the pH in a diffusion chamber in a non-invasive way using
the pH dependent fluorescent dye carboxy SNARF-1.
It was found that a pH gradient could be measured in a cells/agarose mixture in a
diffusion chamber. Although the shape of the measured gradients was not always symmetrical
and the calibration has to be improved slightly, the method could be used for measuring pH
gradients in constructs in a non-invasive and real time way.
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Chapter 1: Introduction
1.1 Articular cartilage
Articular cartilage forms a thin tissue layer that lines the articulating ends of all
diarthrodial joints in the body. The primary functions of this cartilage layer are to minimise
contact stresses generated during joint loading and to contribute to lubrication mechanisms in
the joint. The ability of cartilage to withstand these compressive, tensile, and shear forces
depends on the composition and structural integrity of its extracellular matrix (ECM).
Articular cartilage is composed mainly of water (70-80% by wet weight). It contains cells
(chondrocytes) and collagen fibres embedded in a hydrophilic gel consisting of various
disaccharide polymer chains, called glycosaminoglycans (GAGs). In healthy articular
cartilage the wet weight fraction of GAG is 7% [1]. The most abundant GAGs found in
articular cartilage include chondroitin sulphate, dermatan sulphate, keratan sulphate, and
hyarulonic acid. These GAG molecules are usually present in association with a protein
molecule, forming large aggregated macromolecules called proteoglycans (PGs) which have
the appearance of a bottle brush. The proteoglycan monomers are attached to the hyaluronic
acid chain to form an amorphous gel. In a hydrated environment at physiological pH, the
sulphate and carboxyl groups of the GAG molecules aggregated in proteoglycans are
negatively charged. These negative charges attract cations, which in turn bring in water to
minimise differences in osmotic pressure. The tension in the network of collagen fibres rises
to resist the swelling pressure generated by the proteoglycans [2].
Chondrocytes are small (10 m in diameter), round cells and in most cases
cytoplasmically isolated from their neighbouring cells. They have no ready access to the
vascular system and the tissue is not innervated. The main function of the chondrocytes is to
synthesise, remodel and turnover the ECM.
1.2 Metabolism and gradients in cartilage
In the typical mammalian cell (aerobe conditions), carbohydrates are broken down
through the Embden-Meyerhof-Parnas pathway. In this pathway glucose is converted to
pyruvate, which is converted to acetyl-coenzyme A. Acetyl-coenzyme A enters the Krebs
cycle, in which the remaining carbon is liberated as CO2. Articular cartilage is characterised
by a low O2 consumption and minimal release of CO2. Carbohydrate breakdown in this tissue
is dominated by a near-quantitative conversion of glucose to lactate as an end-product of
glycolysis [3, 4, 5], a reaction sequence which consumes no O2. This anaerobic pathway is
preferentially maintained even under aerobic conditions [6].
As cartilage is avascular, nutrients such as oxygen and glucose have to diffuse from the
synovial fluid through the cartilage matrix to the cells. Lactic acid, which is produced by the
cells, is removed through the matrix by the reverse route. The oxygen tension in the synovial
fluid is reported to be 4-10% [7]. Due to oxygen consumption of the chondrocytes and the
diffusion of the O2, the pO2 has been estimated to be as low as 1% in the deep layers [8, 9]. In
addition, lactate concentrations in synovial fluid are reported to be 5-8 mM [10] compared to
1mM in plasma and would be expected to be considerably higher in the depths of the tissue,
causing a low pH. Thus, cells in the deep zone are under low O2 and glucose concentrations
and low pH compared to the surface cells.
1.3 Culture systems
The most common way of studying the metabolism and viability of articular
chondrocytes is by embedding them in a gel. In such a gel the chondrocytes will keep their
round shape, retain their chondrogenic phenotype for long periods of culture [11] and form a
cartilaginous pericellular matrix resembling that formed in vivo. Two regularly used gels are
alginate and agarose. Alginate is a linear co-polymer of L-guluronic and D-mannuronic acid
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residues derived from brown algae. The unique properties of alginates stem from their ability
to form hydrogels in the presence of divalent ions such as calcium and barium and to be
resolubilised in the presence of sodium ions or calcium chelators [12]. This feature is
important, because the embedded chondrocytes can be easily released from the alginate gel
for analysis of for example the viability.
Agarose, which is extracted form seaweed, is a polymer of mainly uncharged
disaccharide units, with a variable number of methoxy groups. The gel forms a three-
dimensional network below 30C, which is thought to be stabilised by hydrogen bonding
between the water molecules and the hydroxyl groups of the galactose residues [13].
Chondrocytes cannot be removed from agarose as easily as from alginate, but agarose has the
advantage over alginate that it starts to gel at a low temperature without addition of a specific
solution.
Horner and Urban studied the metabolic processes and nutrient supplies of isolated
bovine nucleus cells, which were cultured in agarose gels in a diffusion chamber up to 13
days [14]. Horner and Urban hypothesised that the cells in the middle of the construct died,
when the glucose level fell below a critical level or if the pH became to low. In this diffusion
chamber the pH could not be measured easily, so it was difficult to separate the influence of
the glucose depletion and the low pH on the viability of the cells. The same system is used for
studying the behaviour of articular chondrocytes.
1.4 Aim and outline
Chondrocytes in cartilage normally live under low oxygen and low pH. The low pH is
caused by the production of lactic acid by the chondrocytes themselves. It is hypothesised that
due to the negative charges of the GAGs the concentration of H+ ions will be higher and the
pH will thus be lower than in other tissues. In order to investigate this a system was set up
containing a highly negative solution in a dialysis sack. This solution was dialysed by a
neutral solution containing different concentrations of lactic acid. This was left to equilibrate
and the pH in both solutions was measured to see if there was a difference due to the negative
charges.
The main goal of the research project was to investigate the influence of pH on the
viability and metabolism of the chondrocytes, on top of the influence of different conditions
of low glucose and low O2. Chondrocytes in alginate beads were cultured for three days under
different conditions of glucose, O2 and pH and the viability of the cells was monitored during
that period. Samples were taken and assayed for lactate to get a measure for the metabolism
of the cells.
Diffusion chambers can be used for studying metabolic processes of cells. There is
expected to be a pH gradient in the chamber, but it is difficult to measure the pH in those
chambers, because of the glass plates. Therefore the second main goal of this project was to
set up a method to measure the pH in a diffusion chamber in a non-invasive way. It was
chosen to use pH dependent fluorescent dye and measure the pH with a confocal microscope.
A diffusion chamber was build and the dye was calibrated for this system. The chamber was
filled with a cells/agarose mixture and the pH gradient was measured. Some images were also
analysed to investigate if the same method could be used for measuring a local pH gradient
around one cell.
Because it is difficult to measure the pH in a diffusion chamber, it is also difficult to
validate the new measuring method. In order to do this a numerical model is derived which
predicts the lactic acid gradient in the chamber by modelling the lactic acid production of the
cells and the diffusion of lactic acid through the construct.
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Chapter 2: Materials and Methods

2.1 Materials
The chemicals that were used are listed in table 2.1. In this report the abbreviations
DMEM1, DMEM2, DMEM3, DMEM4 will be used for the following media:
DMEM1: Dulbeccos Modified Eagles Medium (Sigma D5523) supplemented with 44 mM

HEPES, and 0.1% (vol/vol) sodium azide
DMEM2: Dulbeccos Modified Eagles Medium (Invitrogen) supplemented with 1%
(vol/vol) antibiotic/antimycotic solution
HEPES, 1% (vol/vol) antibiotic/antimycotic solution, and 44 mM sodium chloride.
HEPES, 1% (vol/vol) antibiotic/antimycotic solution, and 44 mM sodium chloride.
Table 2.1: Chemicals used

Chemical Catalogue number and supplier
Agarose A9045, Sigma-Aldrich Company Ltd, Poole UK
Antibiotic/antimycotic solution 15240-062, Invitrogen Ltd, Paisley UK
Carboxy SNARF-1 C-1270, Molecular Probes, Leiden The
Netherlands
Chondroitin Sulfate A C8529, Sigma-Aldrich Company Ltd, Poole UK
Collagenase C0130, Sigma-Aldrich Company Ltd, Poole UK
Dulbeccos Modified Eagles Medium 223020-022, Invitrogen Ltd, Paisley UK
(with 25 mM HEPES, sodium pyruvate, 1000mg
l-1 glucose, and pyridoxine)
Dulbeccos Modified Eagles Medium D2902, Sigma-Aldrich Company Ltd, Poole UK
(with L-glutamine, 1000 mg l-1 glucose, without
phenol red and sodium bicarbonate)
Dulbeccos Modified Eagles Medium (without D5030, Sigma-Aldrich Company Ltd, Poole UK
L-glutamine, glucose, phenol red, sodium
pyruvate and sodium bicarbonate)
Dulbeccos Modified Eagles Medium D5523, Sigma-Aldrich Company Ltd, Poole UK
(with L-glutamine and 1000 mg l-1 glucose.
Without sodium bicarbonate)
HEPES H3375, Sigma-Aldrich Company Ltd, Poole UK
L-(+)-Lactic Acid L-1875, Sigma-Aldrich Company Ltd, Poole UK
Lactate reagent 735-10, Sigma-Aldrich Company Ltd, Poole UK
Phosphorus pentoxide AC 200890010 Fisher Scientific UK Limited
Polyethylene glycol 35000 81310 Fluka and Riedel-de Han
Sterile wells plates Falcon, Fahrenheit Laboratory Supplies, Milton
Keynes, UK
Sodium Azide 26628-22-8, VWR International
2.2 Lactic acid
2.2.1 Effect of lactic acid on pH in cartilage

In cartilage the swelling pressure generated by the negatively charged GAGs is in
equilibrium with tension in the collagen fibres. It is hypothesised that due to the negative
charges, the concentration of H+ ions will be higher than in normal tissue and the pH will thus
be lower.
To validate this hypothesis a model system was used, which consisted of a dialysis sack
filled with negatively charged chondroitin sulphate (CS) solved in DMEM1. This sack is
placed in a tube with neutral DMEM1 solution containing polyethylene glycol (PEG).
Different concentrations of lactic acid were added to vary the pH of the PEG solution.
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The tube was placed on a roller bench and left for 72 hours to reach an osmotic
equilibrium. The dialysis sack had a molecular cut of weight of 3.5 kD, meaning that the CS
and PEG molecules (10 kD and 35 kD respectively) could not pass the sack and only the
small molecules could move to equilibrate the osmotic pressures of the PEG and CS
solutions. Since the CS is expected to attract H+-ions, the pH will be lower in the sack than
outside. When the PEG concentration in the tube is increased, more water will be drawn from
the sack and the final CS concentration inside the sack will be higher and thus the pH
difference between the inside and the outside of the sack is expected to be higher.
After the 72 hours, the dialysis sack was removed from the tube, the pH of the PEG
solution was measured with a pH sensor and 1 ml of this solution was pipetted in a pre-
weighed petri dish. Then, one of the clips of the sack was removed and the pH of the CS
solution was also measured, by placing the pH sensor in the dialysis sack. The CS solution
was pipetted in a pre-weighed petri dish. All the petri dishes were weighed again to get the
wet weight of the samples. The first series of samples was dried in an oven at 65 C for a few
days. It was thought that the samples did not dry completely in the oven, therefore the
following series of samples were placed in a dessicator with at the bottom a glass petri dish
with phosphor pentoxide. This phosphor pentoxide would attract water and form phosphoric
acid, thus drying the samples. After a few days the phosphoric acid was removed and new
phosphor pentoxide was added to remove the last water from the samples. When the samples
were dry they were weighted again to determine the dry weight. By dividing the dry weight of
the samples by their wet weight, the final PEG and CS concentrations could be calculated. All
experiments were done in triplicate.
2.2.2 Influence of PEG on pH

PEG is reported to induce an excluded volume effect [15]. This effect arises when large
molecules are dissolved in a solution. The large molecules will take up a large amount of
volume, causing the local concentrations of small molecules in the solution to rise. This
means that the concentration of H+ ions will also rise, causing the pH to lower. To see
whether this was the case in the used DMEM1 two experiments were carried out. First,
DMEM1 with and without 10% PEG was titrated with 0.1 M HCl to see if the buffer capacity
of DMEM1 changed due to the PEG. Second, different amounts of PEG were added to a 0.15
M saline solution and to DMEM1 to see if the PEG concentration would influence the pH.
The pH was measured at 0, 5, 10, 15, 20 and 25% w/v PEG.
2.3 Chondrocytes
2.3.1 Tissue dissection

Feet from 18-24 months old steers were obtained from a local abattoir within 2-3 hours
of slaughter. Articular chondrocytes were isolated from the metacarpal-phalangeal joint. The
feet were cleaned thoroughly, skinned and then the hoof was removed. The joint was
submerged in a sodium hypochlorite solution (10,000 ppm) for at least 30 mins. The joint was
then placed in a class I laminar flow hood. The joint was exposed and cartilage was carefully
scraped off and placed in a 50 ml tube containing DMEM2 with 1 mg ml-1 collagenase.
2.3.2 Tissue digestion

The dissected tissues were enzymatically digested at 37 C in DMEM2 containing 1 mg
ml-1 collagenase in an incubator under 95% Air and 5% CO2 for approximately 18 hours.
After incubation the digested tissue suspension was filtered to isolate the cells. This was done
under aseptic conditions by filtering the digested tissue first through a coarse filter to remove
undigested tissue and then through a 20 m-pore cell strainer. The cells in the filtrate were
then washed three times with DMEM2 by repeated centrifugation in an ALC PK130
centrifuge (2500 rpm for 5 mins) and resuspended in DMEM2. The cells were then counted
and prepared for culture.
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2.3.3 Cell counting

The cell viability and cell number of the cell suspension were determined
microscopically using a trypan blue staining. A 50 l sample of the cell suspension was
diluted with 100 l of 0.4% (w/v) trypan blue in Phosphate Buffered Saline. The suspension
was then loaded onto a Neubauer Improved haemocytometer and manually counted for living
(clear) and dead (blue cells). Multiple counts were performed for each cell preparation and the
number of cells and the cell viability calculated. Only cell preparations with cell viabilities >
95% were then used.
2.3.4 Alginate beads

Cells were encapsulated in alginate beads as described by Guo et al [16]. Alginate
solution (1.2% w/v) was prepared by adding 240 mg sodium alginate to 20 ml 0.9% NaCl and
stirring rapidly for at least 2 hours on a magnetic stirrer. This solution was then sterile filtered
through a 20 m filter (Millipore, UK). After centrifuging (2500 rpm 5 mins), the cells were
resuspended uniformly into 1.2% low viscosity alginate at a density of 4106 cells ml-1 by
gently pipetting, making sure no air bubbles were trapped in the solution. Droplets were
formed by gently expressing the cell suspension through a 21-G needle attached to a 5 ml or
10 ml syringe into a 102 mM CaCl2 solution. The beads were washed three times with 25 ml
of 0.9% NaCl solution and washed twice with 25 ml DMEM2.
2.3.5 Releasing cells from beads

Alginate beads were dissolved in a citrate buffer (1.6% w/v sodium citrate, 1.8% w/v
EDTA, 0.9% w/v sodium chloride). This was left for 5-10 minutes, with gentle agitation. This
solution could be used directly for cell counting. When the cells were needed for other
purposes the cells were then washed two times with DMEM2 by repeated centrifugation
(2500 rpm 5 mins).
2.4 Viability studies
2.4.1 Viability experiment 1

Chondrocytes in cartilage are reported to survive under low O2 concentrations and low
pH values [8, 9, 10]. An experiment was done to find out how the viability of chondrocytes
was influenced by different O2 and pH culture conditions.
After initial incubation of 4 days (in DMEM2 supplemented with 6% FBS), groups of 5
beads were transferred to the wells of sterile plates. DMEM3 was made to either a pH of 7.4
or 6.2. This was added to the wells to a concentration of 0.2106 cells ml-1 medium. For each
pH value, one plate was cultured at 37o C and 100% humidity, in an incubator at 21% oxygen.
Another plate was placed in a sealed vessel (tested at low oxygen concentrations and found to
be airtight) and flushed exhaustively with oxygen-free gas. The concentration of oxygen in
culture plate wells inside the vessel has been tested and was found to decrease to that of the
gas mixture flushed through the chamber. The remainder of the gas comprised 5%
CO2/balance N2. This experimental protocol gave 4 different combinations of pH and oxygen
concentration; each was done in triplicate. After 18, 42 and 66 hours all beads were removed
from each well, dissolved in a citrate buffer and the cell viability was assayed.

Glucose is the most important nutrient for chondrocytes. Therefore in addition to the
different culture conditions of O2 and pH from the first viability experiment the glucose
concentration was varied.
In this experiment beads were used after one day of initial incubation. DMEM3 (5mM
glucose) and DMEM4 (no-glucose) were made to either a pH of 7.4 or 6.2. For each value of
glucose and pH, one plate was cultured at 21% oxygen and another plate at 0% oxygen. (see
2.4.1). This experimental protocol gave 8 different combinations of glucose, pH and oxygen
concentration; each was done in triplicate.
8

The goal of this experiment was to repeat viability experiment 2 to see whether the
chondrocytes from another animal responded in the same way to the different culture
conditions of pH, O2 and glucose concentrations. Unfortunately, the cartilage pieces had to be
placed in DMEM2 supplemented with 6% FBS for 48 hours, before starting tissue digestion
with collagenase. After the digestion the alginate beads could be made.
The beads were supposed to be cultured under the same 8 conditions as in viability
experiment 2. But the vessel appeared to have a leak and was not airtight. The O2
concentration in the medium after flushing started at 0%, but due to the leak it rose to 10%
in 24 hours. Because every day one plate was removed from the vessel for counting, it had to
be gassed again. This gave a fluctuating O2 concentration from 0% to 10% per day.
2.4.4 Lactate assay

During the viability experiments the chondrocytes metabolised glucose, which was
converted into lactate. To see whether the different culture conditions influenced this
metabolic process, the lactate concentration in the medium was determined at each time point.
This was done by performing a lactate assay at 540 nm using a lactate reagent on a sample of
medium.
Because during the experiment some of the cells died, the lactate concentrations had to
be normalised for the amount of living cells, which contributed to the measured lactate
concentration. The average number of living cells was calculated using the total number of
cells and the number of live cells. (Equation 1)
total number of cells (t = 0) + number of living cells (t = x)

= average number of cells (1)
2
This average number was used to calculate the amount of lactate produced per million cells.
(Equation 2)
1 10 6
Amount of lactate = amount of lactate per million cells (2)
average number of cells
2.5 pH gradient in diffusion chamber
Diffusion chambers can be used to investigate the influence of O2, lactic acid and
glucose gradients on the metabolism and viability of chondrocytes [14]. The cells in the
centre of those chambers can only get their nutrients through to diffusion and their waste
products can only be removed through diffusion. This means that the lactic acid concentration
in the centre of the chamber is higher than at the edges and that a symmetrical pH gradient
will from across the chamber.
The goal of this part of the study was to set up a method to measure such a pH gradient
using a pH dependent fluorescent dye.
2.5.1 Experimental set-up

The design of the diffusion chambers, modified from that described by [14], consisted of
two impervious glass plates, a circular one with a diameter of 35 mm and a square one of 24 x
24 mm. These plates were held apart on two sides by 170-m spacers, resulting in a chamber
with 2 closed sides (figure 2.1). The spacers have a width of 2 mm, which makes the effective
size of the diffusion chamber 24 x 20 x 0.170 mm. The diffusion chamber was glued in a
special designed petri dish with low modulus silicone sealant to give a watertight seal and left
to dry overnight. The petri dish was made to minimise the distance from the objective of the
microscope to the middle of the chamber and was designed to fit on a specific stage of the
Bio-Rad Radiance 2000 confocal microscope. To minimise the amount of dye a ring could be
placed in the petri dish. The diffusion chamber and the petri dish are shown in figure 2.1.
9
After the glue had dried the diffusion chamber was placed in alcohol for 5-15 minutes for
sterilisation. When the chamber was dry, it was ready to be filled with the agarose and cell
suspension.
Petri dish
Nutrients diffuse
from the medium Removable ring
into the open sides
Spacer separating
the upper and lower
glass plates
Figure 2.1: A schematic view of the diffusion chamber in the petri dish, side and top view
2.5.2 Carboxy SNARF-1 pH indicator

Carboxy SNARF-1 is a long-wavelength fluorescent pH indicator developed by
Molecular Probes. The absorption spectrum of the carboxy SNARF-1 undergoes a shift to
longer wavelengths upon deprotonation of its phenolic substituent. Carboxy SNARF-1 also
exhibits a significant pH-dependent emission shift from yellow-orange to deep red
fluorescence from acidic to basic conditions, respectively. This pH dependence allows the
ratio of the fluorescence intensities from the dye at two emission wavelengths, 580 nm and
640 nm, to be used for quantitative determinations of pH [17]. The pH-dependent emission
spectra of carboxy SNARF-1 when it is excited at 514 nm is shown in figure 2.2.
Figure 2.2: Emission spectra carboxy SNARF-1
2.5.3 Determination of the right concentration of dye

For the determination of the right concentration of dye, a thin layer of agarose was
pipetted on a coverglass, which was glued on the petri dish. The petri dish was placed in a
fridge for 15-30 minutes to allow the agarose to gel. Meanwhile three different concentrations
(5, 10, 25 M) of Carboxy SNARF-1 were made up in DMEM3 (without phenol-red and
without bicarbonate). Phenol-red is a pH sensitive dye normally present in DMEM. In this
experiment phenol red was omitted, because it could give extra background noise.
Bicarbonate is the main buffer present in DMEM. This buffer only works properly when the
DMEM is placed under 95% Air and 5% CO2. If DMEM with this buffer is placed in normal
air, which has a very low CO2 concentration, it will loose all its CO2 and the pH of the
DMEM will rise. The experiments were all carried out on a microscope without a CO2
incubation system and therefore DMEM3 without bicarbonate was used. To replace the
10
bicarbonate buffer 25 mM of HEPES was added. NaCl was added to get an osmolarity of 380
mOsm. The petri dish was taken out of the fridge and 4 ml of the DMEM3 with the lowest
Carboxy SNARF-1 concentration was added. This was left to equilibrate for 15 minutes and
thereafter the intensity of the signal was measured. Then the medium was removed and 4 ml
of the medium with 10 M Carboxy SNARF-1 was added. This was left for 15 minutes to
equilibrate and the intensity measurements were repeated. This was also done for the 25 M
concentration.
The 25 M concentration appeared to give a good enough signal to noise ratio to
measure a pH gradient across the diffusion chamber. When measuring closer around the cells
a higher concentration is needed [18, 19].
2.5.4 Calibration curve

The ratio of the Carboxy SNARF-1 emission intensities depend on many factors and
therefore a calibration curve had to be made for this specific experimental set up. This was
done using the same kind of agarose layer as for the determination of the concentration and
was done in the same way. This experiment the DMEM solutions all had the determined
Carboxy SNARF-1 concentration (25 M), but the pH values ranged from 7.43 to 6.3. At the
beginning and at the end of the measurements a blank measurement was taken.
The ratio of each pH value was determined by taking the intensity at 480 nm minus its blank,
divided by the intensity at 540 nm minus its blank (equation 3). Figure 2.3 shows the
calibration curve.
I I
Ratio = 480 480blank (3)
I 540 I 540blank
7.6
7.4
7.2
y = -2.523x + 8.3476
7
pH (-)
6.8
6.6
6.4
6.2
0 0.2 0.4 0.6 0.8 1
Ratio (-)
Figure 2.3: Calibration curve
2.5.5 pH gradient experiments

To see if a pH gradient along a diffusion chamber could be measured with the described
method some experiments were carried out. First off all the chambers were filled with a
cells/agarose (4106 cells ml-1 and 1% agarose) mixture and put in the fridge for 15-30 minutes
to gel. Then DMEM3 was added to the chamber. The DMEM3 was changed with DMEM3
with 100 M carboxy SNARF-1 18 hours prior to the measurements. The measurements were
carried out 24 or 72 hours after the chambers were filled with the cells/agarose mixture. After
the measurements the pH of the medium was measured with a pH electrode.
A pilot measurement was carried out to see if the method worked. The next chambers
were cultured with either DMEM3 with the usual glucose concentration (5 mM) or with a
higher glucose concentration (10 mM). At a glucose concentration of 5 mM the cells in the
centre of the chamber normally die within 3 days, because they run out of glucose [14]. When
the glucose concentration is doubled to 10 mM the cells have enough glucose to survive.
Because more cells stay alive in the centre, there could be differences in the amount of lactic
acid that is produced in the centre of the chamber, which of course could make a difference in
the pH gradient.
11
2.5.6 pH gradient around one cell

The global pH gradient in a diffusion chamber is caused by the lactic acid concentration.
Because the cell is the producer of the lactic acid, it is expected that there also is a local pH
gradient around the cell. The hypothesis is that closer to the cell the pH will be lower than
further away from the cell.
After the global measurements of section 2.5.5 the microscope was zoomed in to obtain
images with only one or two cell in the field of view. Again both emission intensities were
measured and from the ratio the pH could be determined. Circles, of which the mean intensity
was calculated, were drawn around the cells (figure 2.4). Due to the shape of the regions, the
intensity from the cell was also taken into account when calculating the mean intensity of that
region. To calculate the mean of the area around the cell, each time two adjoining circles were
taken and the mean intensity of the smaller circle multiplied by its area was subtracted from
the mean intensity multiplied by the area of the larger one. In addition, a large square region
further away from the cell was also analysed to determine a reference value.
Figure 2.4: Circles around cell for analysis of pH gradient around one cell.
Also one image containing two cells was analysed. It was expected that the difference in
pH would be larger, because there were now two cells producing lactic acid in a small area.
This time, regions were drawn without including the cells. So the mean values of intensity of
the region could be used directly for calculating the ratio.
2.6 Numerical model lactic acid production
In section 2.5 of this report a method was described for measuring a pH gradient in a
diffusion chamber. There were no other ways to measure the pH within such a chamber, so it
was difficult to verify whether the measured values were correct. Because the pH gradient is
mainly caused by the production of lactic acid, an attempt was made to predict the pH
gradients with a numerical model based on the production of lactic acid by the cells and its
diffusion through the agarose out of the chamber. Both processes are included in the model as
simple 1D equations, because the diffusion chamber is a square symmetrical set up and the
cells/agarose mixture is assumed homogeneous. It is assumed that the cells do not divide, so
the cell density is assumed to be constant. The production of lactic acid is assumed to be a
constant value per cell and the diffusion is modelled with the normal diffusion equation. This
results in the combined equation:
C 2C
=D + R, with R = Rbasic Ccell (5)
t x 2
12
With:
t = Time
x = Position
C = Lactic acid concentration (mol mm-3)
D = The diffusion coefficient (mm s-1)
R = Production rate lactic acid (mol mm-3 s-1)
Rbasic = Basic production rate lactic acid (mol cells-1 s-1)
Ccell = Concentration of cells (cells mm-3)
The lactic acid production over 24 hours was modelled. The cell density was set at 4106 cells
ml-1 and the diffusion coefficient for lactic acid through agarose was set at 910-4 mm2 s-1 [20].
The lactic acid production rate per cell was estimated from the lactate measurements carried
out in viability experiment 2 and was set at 4.8210-11 mol cell-1 s-1.
13
Chapter 3: Results
3.1 Lactic acid
3.1.1 Effect of lactic acid on pH in cartilage

In this experiment the pH was measured in PEG and CS solutions that were left to
equilibrate for 72 hours at different lactic acid concentrations. The results are shown in figure
3.1. The experiments with the 10% PEG solution show that for a lactic acid concentration
between 0 and 15 mM the pH of the CS is slightly lower than in the PEG solution itself. For
higher lactic acid concentrations, the pH of the CS is slightly higher than the pH of the PEG
solution. When using 10% PEG, the final concentration of CS was 8.30 0.23 (mean +
SEM). When the PEG concentration is increased to 15%, the final CS concentration was
12.45 0.18 (mean + SEM) and the difference of the pH between PEG and CS solution
increased.
8
PEG (10% PEG)
CS (10% PEG)
7.5 PEG (15% PEG)
CS (15% PEG)
7
6.5
pH (-)
5.5
4.5
4
0 5 10 15 20 25 30
Lactic acid (mM)
Figure 3.1: pH of PEG and CS solutions after 72 hours at different lactic acid concentrations
The final concentrations of CS versus the final concentrations of PEG are shown in
figure 3.2. When the PEG concentration is higher, the CS concentration is also higher. The
samples of one series have a large spread. This is not to be expected, because this kind of
equilibrium should reach exactly the same end point, when starting with the same
concentrations. The final CS concentrations of the first series are clearly lower than the CS
concentrations of the other series. Although phosphor pentoxide is often used for drying
samples it seems that these samples dried better in the oven.
14
Series1
Series2
13 Series3
Series4
12 Series5
11
CS (% wt)
10
6
10 11 12 13 14 15 16 17
PEG (% wt)
Figure 3.2: CS concentration versus PEG concentration. Series 1 was dried in the oven and series 2-5 are dried
using the phosphor pentoxide.
14
3.1.2 Influence of PEG on pH

The titration curves of DMEM1 with or without 10% PEG have the same shape (figure
3.3A) meaning that the buffer capacity of the DMEM1 isnt altered by the PEG. The pH at
different concentrations of PEG in a saline solution and DMEM1 are shown in figure 3.3B.
The pH of the saline solution drops as the PEG concentration increases. The pH of the
DMEM1 on the other hand is not influenced by the PEG concentration.
8 8
DMEM+10%PEG Saline
7.5 DMEM
DMEM 7.5
7
6.5
7
6
pH (-)
pH (-)
5.5 6.5
5
6
4.5
4
5.5
3.5
3 5
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25
HCl (mM) PEG (% wt)
A B
Figure 3.3: A: Titration curves of DMEM with and without 10% PEG. B: pH of saline solution and DMEM versus
PEG concentration
3.2 Viability studies

The viability profiles are shown in figure 3.4A. The cells stay alive for two days at
pH=7.4 and 21% O2, after that the viability lowers. Cells cultured at pH=7.4 and 0% O2 have
a lower viability. All the cells cultured at pH=6.2 show a lower viability than at pH=7.4. If the
cells are cultured at 0% O2 the viability is even lower.
The lactate production of the cells is higher at pH=7.4 than at pH=6.2, there does not
seem to be a difference between 21% O2 and 0% O2 (figure 3.4B).
100 45
40
80
Amount lactate produced
35
(umol/1e6 cells)
30
Viability (%)
60
25
20
40
15
20 10
5
0 0
0 1 2 3 0 1 2 3
Time (days) Time (days)
+O2 pH=7.4 +O2 pH=6.2 +O2 pH=6.2 -O2 pH=7.4
-O2 pH=7.4 -O2 pH=6.2 -O2 pH=6.2 +O2 pH=7.4
A B
Figure 3.4: A: Cell viability versus time. B: Lactate production of cells versus time.

In addition to pH and O2, the glucose concentration was varied (5 mM or 0 mM). When
the glucose concentration is 5 mM, it can be seen that the cells stay alive at pH=7.4. The cells
cultured at pH=6.2 are less viable. The O2 concentration does not seem to make a difference.
When the glucose concentration is 0 mM, cells start to die in every condition. The cells
cultured at pH=6.2 have a lower viability than the ones at pH=7.4. The viability at 0% O2 is
even lower.
The amount of lactate produced by the cells is highest under high glucose, pH=7.4 and
21% O2. It is lower at 0% O2, but between day 2 and 3 the cells still produce lactate. At high
glucose, pH=6.2 and 21% O2 a rise is visible in the produced amount of lactate for all three
15
days. The other conditions do not show this rise as clearly; most of them have the same
amount of lactate at day 2 and 3 meaning that there is no lactate produced.
16
100
14

80 12
(umol/1e6 cells)
Viability (%)
10
60
8
40 6
4
20
2
0 0
0 1 2 3 0 1 2 3
+O2 High Gluc pH=7.4 +O2 No Gluc pH=7.4 +O2 High Gluc pH=7.4 +O2 No Gluc pH=7.4
-O2 High Gluc pH=7.4 -O2 No Gluc pH=7.4 -O2 High Gluc pH=7.4 -O2 No Gluc pH=7.4
A B

This experiment was performed under the same conditions as experiment 2 (3.2.2), only
in this experiment the low O2 concentrations was 0-10%. The cells came from an older foot
and the cartilage was stored for 48 hours in DMEM2 before starting the digestion of the
tissue. The results for the 5 mM glucose concentration were the same as for viability
experiment 2. The only thing that was different was the influence of the 0-10% O2
concentration at the 0 mM glucose concentration. At this concentration the viability was
higher than the 21% O2.
The amount of lactate produced by the cells is highest under high glucose, pH=7.4 and
21% O2. It is lower at 0% O2, the sudden drop at day three is likely to be an error in the
measurements, because the viability of the cells is almost 100% and the samples taken at day
1 and day 2 show a much higher amount of lactate present. The other conditions show a much
lower amount of lactate again most of them do not show a rise between day 2 and 3.
16
100
14
80 12
(umol/1e6 cells)
Viability (%)
10
60
8
40 6
4
20
2
0 0
0 1 2 3 0 1 2 3
A B
3.3 pH gradient in sandwich
The filling of the agarose / cell mixture had to

be pipetted very quick into chamber, because the
agarose would start to gel. A petri dish with a
chamber filled with the agarose / cell mixture is
shown in figure 3.7. This chamber is filled from
the right side. The agarose has spread perfectly to
the left side of the chamber. The right side shows
some air bubbles.
Figure 3.7: Petri dish with chamber filled with agarose.
16
3.3.1 pH gradient experiments

The first pH gradient experiment that was carried out gave a clear gradient in the ratio
between the two signals (figure 3.8). These ratio values were converted to pH values (figure
3.9), using the calibration curve (figure 2.3). There was a gradient present, but the peak of the
gradient did not lye in the middle of the sample. The pH of the medium around the chamber
was 7.28.
0.74 6.8
0.72 6.75
6.7
0.7
6.65
Ratio (-)
pH (-)
0.68
6.6
0.66
6.55
0.64 6.5
0.62 6.45
0 5 10 15 20 25 0 5 10 15 20 25
Distance (mm) Distance (mm)
Figure 3.8: Ratios in diffusion chamber Figure 3.9 Calculated pH in diffusion chamber
The gradients of the chambers after 24 hours with 5 mM glucose and 10 mM glucose are
shown in figure 3.10A and 3.10B respectively. The gradient in figure 3.10A is non-
symmetrical. The gradient in figure 3.10B looks better, but the graph is again not
symmetrical. Because of the strange shapes of the gradients it is difficult to say whether the
glucose concentration made a difference. The pH of the medium around the chamber was 7.3.
7 7
6.95 6.95
6.9 6.9
6.85 6.85
6.8 6.8
pH (-)
pH (-)
6.75 6.75
6.7 6.7
6.65 6.65
6.6 6.6
6.55 6.55
0 5 10 15 20 25 0 5 10 15 20 25
A B
Figure 3.10: pH gradients across diffusion chamber after 24 hours, with 5 mM glucose (A) and 10 mM glucose (B)
The gradients of the chambers after 72 hours with 5 mM glucose and 10 mM glucose are
shown in figure 3.11A and 3.11B respectively. Both gradients do not look very nice. The
figures also show that it was not possible to measure the complete 24 mm of the chamber.
The pH of the medium around the chamber was 7.3.
7.25 7.25
7.2 7.2
7.15 7.15
7.1 7.1
pH (-)
pH (-)
7.05 7.05
7 7
6.95 6.95
6.9 6.9
6.85 6.85
0 5 10 15 20 25 0 5 10 15 20 25
A B
Figure 3.11: pH gradients across diffusion chamber after 72 hours, with 5mM glucose (A) and 10mM glucose (B)
17
3.3.2 pH gradient around one cell

Two cells with their regions analysed for a local pH gradient are shown in figures 3.12A
and 3.13A The region numbers start at the edge of the cell and go outward till the last
number, which is the reference square. Figures 3.12B and 3.13B show the calculated pHs. It
can be seen that there are no significant differences in pH between the regions around one
cell, so no gradient can measured.
6.3
6.25
pH (-)
6.2
6.15
6.1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Regions
A B
Figure 3.12: A: Regions used for calculation. B: Calculated pHs
6.3
6.25
pH (-)
6.2
6.15
6.1
0 1 2 3 4 5 6 7 8 9 10
Region
A B
The regions used for measuring the pH around two cells are shown in figure 3.14A.
Region number 1 is the region between the two cells. Region numbers 2 6 are the regions
going away from the cells and region number 7 is the reference square. Again no significant
differences can be seen between the regions.
6.3
6.25
pH (-)
6.2
6.15
6.1
0 1 2 3 4 5 6 7 8
Region
A B
18
3.4 Numerical model lactic acid production
A numerical model was used to predict the lactic acid concentrations in an agarose construct
for 24 hours. The result of the simulation is shown in figure 3.15. It can be seen that the lactic
acid concentration within the construct and rises to 11 mM in the centre of the construct (at 13
mm).
Lactic acid concentration
12
t=0
t=2
t=4
t=6
10 t=8
t=10
t=12
t=14
8 t=16
Concentration (mM)
t=18
t=20
t=22
6 t=24
0
0 2 4 6 8 10 12 14
Distance (mm)
Figure 3.15: Lactic acid concentration in agarose construct (lines are drawn every two hours)
19
Discussion
The goal of this report was threefold, first of all the effect from lactic acid on the pH was
investigated using a model system for cartilage. The second aim of the research project was to
investigate the influence of pH on the viability and metabolism of the chondrocytes, on top of
the influence of different conditions of low glucose and low O2. The third goal was to set up a
method to measure the pH gradient in a diffusion chamber in a non-invasive way and predict
the gradient with a numerical model.
To determine the effect of lactic acid on the pH, a model system for cartilage was used in
which a CS solution was equilibrated with a PEG solution. It was found that for lactic acid
concentrations from 0 15 mM the pH of the CS solution was slightly lower than the pH of
the PEG solution. For higher concentrations the pH of the CS solution was slightly higher
than the PEG solution. The difference in pH of the PEG solution and the CS solution was not
significant in most points. This is caused by the fact that the differences were very small and
that the starting pHs of media from different series were not exactly the same. The fact that
at higher lactic acid concentrations the pH of the CS was higher than the pH of the PEG can
be explained by the fact that the pKa value for carboxyl groups of chondroitin sulfate is 3.97
[21]. The H+ ions will start to bond with these groups, thus acting as a buffer. When a higher
PEG concentration was used, the resulting CS concentration was also higher and there was a
bigger difference between the pH of the PEG solution and the pH of the CS solution. When
doing this kind of equilibrium experiments the final CS concentration is totally determined by
the starting concentration of the PEG solution. This means that the CS concentrations of
samples from the same series should end up at the same value. This was not seen in these
experiments and is an indication that the determination of the final concentrations was not
correct. They were determined by dividing the dry weight by the wet weight of the sample. If
the samples are not completely dry the final concentration ends up higher. Thus because
differences were seen in the final concentrations the method of drying should be checked.
When adding PEG to DMEM1 no excluded volume could be found and the buffer
capacity of the medium did not change, meaning that the pH measurements were not
influenced by the PEG solution.
The viability experiments showed that cells cultured without glucose immediately start to
die. On the other hand when the cells are cultured with 5 mM glucose the viability is much
higher. When culturing the cells at pH=7.4 and 21% O2 the viability is almost 100%. Every
time the cells were cultured at pH=6.2 the viability turned out lower than of cells cultured at
pH=7.4.
The influence of O2 was different in all three experiments. The first experiment showed
that the cells cultured with glucose and pH=7.4 under 0% O2 had a lower viability than cells
cultured under 21% O2. In the second and third experiment the oxygen concentration did not
have an effect on the cells when there was glucose present. The cells from the first experiment
were taken from 4 day-old alginate beads and had a start viability of 95%. This was low
compared to the other experiments [22]. It could well be that the cells were not in a perfect
condition and this could explain the different reaction to the O2 concentration.
The viability experiments 2 and 3 showed that cells cultured without glucose and 0% O2
had a lower viability compared to the 21% O2. When the cells were cultured with a low O2
concentration (0-10%), the cells were more viable. It is not clear if the difference in viability
between the 0% O2 and 0-10% O2 concentration is solely caused by this O2 concentration, or
by the different way of harvesting the cells.
A pH gradient could be measured in a cells/agarose mixture in a diffusion chamber using

the pH dependent fluorescent dye carboxy SNARF-1. Yet, there were some problems. First of
the all, the measured gradients were not symmetrical, which they should have been because of
the shape chamber. Another problem was that in some experiments it was not possible to
measure the pH across the total length of the chamber. A possible reason for this was that it
20
was difficult to find the edge of the agarose, because the agarose slightly shrunk during the
drying. Hence, the first measurement was not always done on the edge of the chamber but
already inside the chamber. Another problem was that sometimes the cells/agarose mixture
was slightly spilled when filling the chamber. Because this mixture is not covered by a glass
plate and the H+-ions can easily diffuse in and out of the agarose, the pH is likely to be equal
to the surrounding medium. When measurements are done in this part of the cells/agarose and
interpreted as if they were done inside the chamber the gradient would we shifted.
There were differences between the pH at the edge of the diffusion chamber and the pH
of the surrounding medium. This was not expected, because the H+ ions at the edge should be
able to diffuse rapidly into the medium. Calibration was done in agarose gel without the cover
of the top glass plate that was present in the gradient measurements. Although this was not
expected to make a difference, it could be the cause of the differences between the edge and
the medium. When measuring the last two gradients it was not possible to measure the total
length of the chamber, this was caused by the fact that the lens touched the petri dish.
An attempt was made to measure a local pH gradient around one or two cells. This
gradient was not found. The spread on the signal was too large to measure differences
between the small bands of circles.
The pH at the start of the experiments is 7.4 and drops during the experiments. As
carboxy SNARF-1 indicator has a pKa of 7.5, it is less sensitive for measuring lower pHs.
An alternative dye, with the same single excitation - dual emission properties, but a more
acidic pH sensitive maximum (pKa 6.4) which could be used is carboxy SNARF-4F.
The numerical model predicts a lactic acid gradient within an agarose construct, and can
be easily used for predicting the pH in the chamber. The lactic acid concentrations have to be
coupled with the titration curve of the DMEM to determine the precise pH with the lactic acid
concentration. Some assumptions in the model are important to consider with regard to the
prediction of the pH. First, the cells start to die in the middle of the chamber, possibly because
of the lack of glucose, and will thus stop producing lactate. This was not implemented in the
current model. To minimise the error of this simplification a simulation time of 24 hours was
taken, because the viability experiments showed that after one day most cells are still alive.
Second, the lactic acid production was implemented as a constant. However, the lactic acid
production of cells is known to be dependent on other factors, such as the amount of lactic
acid present.
In conclusion, this study shows that the pH has a clear influence on the viability of
articular chondrocytes, although the effect of the glucose concentration is more pronounced
the effect of a pH change from 7.4 to 6.2. The dye carboxy SNARF-1 can be used for
measuring the pH gradient across a diffusion chamber, but more experiments should be
carried out for the quantification of the pH in such a chamber. Numerical models can help
predict the pH in constructs and therewith assist in interpretation of the experimental data.
21
References
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of glycosaminoglycan distribution in cultured natural and engineered cartilage. Annals of
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2. Shrive NG, Frank CB, Articular Cartilage. In: Biomechanics of the musculo-skeletal
system. Nigg B, Herzog W (ed) J Wiley and Sons, Chichester, 1994
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Rheumatologie 1991; 50:304-312
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enzymes in cultured articular chondrocytes. Proc Soc Exp Biol Med 1973 Jun;
143(2):488-491
7. Ferrel WR, Najafipour H. Changes in synovial pO2 and blood flow in the rabbit knee
joint due to stimulation of the posterior articular nerve. Journal of Physiology 449
(1992):607-617
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energy metabolism. Biorheology 2000; 39:133-143
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Roy Soc London 1975; 271:261-272
10. Kortekangas P, Peltola O, Toivanen A, Aro HT. Synovial fluid L-lactic acid in acute
arthritis of the adult knee joint. Scandinavian Journal of Rheumatology 1995; 24:98-101
11. Hauselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Ayelotte MB, et al.
Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate
beads. Journal Cell Science 1994; 107:17-27
12. Wong M, Siegrist M, Wang X, Hunziker E. Development of mechanically stable

alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis.
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13. Lundberg P, Kuchel PW. Diffusion of Solutes in Agarose and Alginate Gels: 1H and
23Na PFGSE and 23Na TQF NMR Studies. Magnetic Resonance in Medicine 1997;
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14. Horner HA, Urban JPG. 2001 Volvo Award Winner in Basic Science Studies: Effect of
nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral
disc. Spine 2001; 26:2543-2549
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solutions. 1. Mutual diffusion in the ternary system poly(ethylene glycol) 400-NaCl-
Water. Macromolecules 2001; 34:991-1000
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16. Guo JF, Jourdian GW, MacCallum DK. Culture and growth characteristics of
chondrocytes encapsulated in alginate beads. Connective Tissue Research.1989; 19(2-
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17. Molecular Probess Handbook of Fluorescent Probes and Research Products, Ninth
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18. Chu S, Montrose MH. Transepithelial SCFA fluxes link intracellular and extracellular pH
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19. Bassnett S, Reinisch L, Beebe DC. Intracellular pH measurement using single excitation-
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PFGSE and 23Na TQF NMR Studies. Magn. Reson. Med. 1997; 37:44-52
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23

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12

Study of the influence of

Dr. J.P.G. Urban

CHAPTER 1: INTRODUCTION .......................................................................................... 4

1.2 Metabolism and gradients in cartilage

1.3 Culture systems

1.4 Aim and outline

Chapter 2: Materials and Methods

DMEM1: Dulbeccos Modified Eagles Medium (Sigma D5523) supplemented with 44 mM

Table 2.1: Chemicals used

2.2 Lactic acid

2.2.1 Effect of lactic acid on pH in cartilage

2.2.2 Influence of PEG on pH

2.3.1 Tissue dissection

2.3.2 Tissue digestion

2.3.3 Cell counting

2.3.4 Alginate beads

2.3.5 Releasing cells from beads

2.4 Viability studies

2.4.1 Viability experiment 1

2.4.2 Viability experiment 2

2.4.3 Viability experiment 3

2.4.4 Lactate assay

total number of cells (t = 0) + number of living cells (t = x)

2.5 pH gradient in diffusion chamber

2.5.1 Experimental set-up

2.5.2 Carboxy SNARF-1 pH indicator

Figure 2.2: Emission spectra carboxy SNARF-1

2.5.3 Determination of the right concentration of dye

2.5.4 Calibration curve

Figure 2.3: Calibration curve

2.5.5 pH gradient experiments

2.5.6 pH gradient around one cell

2.6 Numerical model lactic acid production

3.1.1 Effect of lactic acid on pH in cartilage

3.1.2 Influence of PEG on pH

3.2 Viability studies

3.2.1 Viability experiment 1

3.2.2 Viability experiment 2

Amount lactate produced

3.2.3 Viability experiment 3

3.3 pH gradient in sandwich

The filling of the agarose / cell mixture had to

3.3.1 pH gradient experiments

3.3.2 pH gradient around one cell

3.4 Numerical model lactic acid production

A pH gradient could be measured in a cells/agarose mixture in a diffusion chamber using

6. Marcus RE, Srivastava VM. Effect of low oxygen tensions on glucose-metabolizing

12. Wong M, Siegrist M, Wang X, Hunziker E. Development of mechanically stable

15. Vergara A, Paduano L, Vitagliano V, Sartorio R. Multicomponent diffusion in crowded

21. Mathews MB. Macromolecular properties of isomeric chondroitin sulphates. Biochimica

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