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Karl Fischer titration and coulometry for measurement of water content in

small cartilage specimens Bestimmung des Wassergehalts in kleinen
Knorpelproben durch Karl-Fischer-Titrati...

Article  in  Biomedizinische Technik · December 2006

DOI: 10.1515/BMT.2006.069

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 Article in press - uncorrected proof
Biomed Tech 2006; 51:xxx-xxx
2006 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BMT.2006.0XX
Karl Fischer titration and coulometry for measurement of
water content in small cartilage specimens
Bestimmung des Wassergehalts in kleinen Knorpelproben durch
Karl-Fischer-Titration und Coulometrie
Gunter Spahn1,*, Holger Plettenberg2, Horst
Nagel3, Enrico Kahl4, Hans Michael Klinger4,
Manfred Günther2, Thomas Mückley5 and
Gunther O. Hofmann5
1
Praxisklinik für Unfallchirurgie und Orthopädie,
Eisenach, Germany
2
fzmb – Forschungszentrum für Medizintechnik und
Biotechnologie, Bad Langensalza, Germany
3
Sartorius AG, Göttingen, Germany
4
Orthopädische Klinik, Georg-August-Universität,
Göttingen, Germany
5
Klinik für Unfall-, Hand- und
Wiederherstellungschirurgie, Friedrich-Schiller-
Universität, Jena, Germany
Abstract
This study evaluated the efficiency of Karl Fischer titra-
tion and coulometry for measurement of water content in
small intact and defective cartilage specimens. Cartilage
from the main weight-bearing zone of the medial condyle
of 38 fresh sheep knees was used. Of these, 20 condyles
had an intact cartilage, while defects (14 grade I and 4
grade II) were found in the rest. The mechanical hardness
was determined as Shore A. Cartilage specimens of
approximately 5 mg were analyzed in special devices for
moisture measurement and then continuously heated up
to 1058C. The actual measurement was performed in an
electric cell (coulometry). An electrode was laminated
with hygroscopic phosphorus pentoxide. In the electro-
chemical reaction, H and O are liberated from the elec-
trode. The requirement for electric energy correlates with
the amount of water in the specimen. The water content
in intact cartilage was 66.9%. Grade I (72.6%) and grade
II (77.8%) defects had significantly higher water content.
Significantly higher and faster spontaneous evaporation
was observed in cartilage defects at room temperature.
The water content and spontaneous water evaporation
correlated with significantly lower mechanical hardness.
The experimental design (combined method of thermo-
gravimetry, Karl Fischer titration, and coulometry) was
sufficient for evaluating the water content in small carti-
lage specimens. It is also possible to measure the tem-
perature-dependent water liberation from cartilage
specimens.
*Corresponding author: Dr. med. Gunter Spahn, Praxisklinik für
Unfallchirurgie und Orthopädie Eisenach, Sophienstr. 16,
99817 Eisenach, Germany
Phone: q49-3691-73500
Fax: q49-3691-735011
E-mail: spahn@pk-eisenach.de
Keywords: cartilage; coulometry; hardness; Karl Fischer
titration; water.
Zusammenfassung
Die Untersuchungen wurden mit dem Ziel durchgeführt,
den Wassergehalt in intaktem und geschädigtem hyali-
nen Knorpel mit der Karl-Fischer-Titration und der Cou-
lometrie zu bestimmen. Knorpelproben wurden aus der
Hauptbelastungszone des medialen Femurkondyls von
38 frischen Schafsknien entnommen. Zwanzig Kondylen
waren intakt, 14-mal wurde ein erstgradiger und 4-mal
ein zweitgradiger Knorpelschaden gefunden. Die mecha-
nische Härtemessung wurde mit dem Shore Härtetester
(Shore A) bestimmt. Danach wurden Proben von ca. 5 mg
entnommen und mit einem speziellen Feuchtigkeitsana-
lysegerät untersucht. Es erfolgte eine kontinuierliche
Erwärmung der Proben von Raumtemperatur bis auf
1058C. Währendessen erfolgte eine kontinuierliche, elek-
trische Messung der Wasserfreisetzung aus den Proben
durch Coulometrie. Dabei wird das freigesetzte Wasser
an einer mit dem extrem hygroskopischen Phosphorpen-
toxid vorbeigeleitet. Durch die elektrochemische Reak-
tion werden H und O aus der Elektrode freigesetzt. Diese
Freisetzung korreliert mit der freigesetzten Wassermen-
ge. Der Wasserhalt in intaktem Knorpel betrug 66,9%. In
erstgradig geschädigtem Knorpel (72,6%) und zweitgra-
dig geschädigtem Knorpel (77,8%) war der Wassergehalt
signifikant höher. In geschädigtem Knorpel fand sich
zudem eine signifikant höhere und schnellere spontane
Verdunstung bei Raumtemperatur. Der erhöhte Wasser-
gehalt und die spontane Verdunstung korrelierten signi-
fikant mit dem Verlust der mechanischen Härte. Das
experimentelle Design (Kombination von Thermogravi-
metrie, Karl-Fischer-Titration und Coulometrie) sind
geeignet zur Wassergehaltsbestimmung in sehr kleinen
Knorpelproben. Dabei ist es zudem möglich, die tempe-
raturabhängige Wasserfreisetzung aus den Knorpelpro-
ben zu messen.
Schlüsselwörter: Coulometrie; Karl-Fischer-Titration;
Härte; Wasser.
Introduction
Physiology and pathophysiology of hyaline cartilage
Cartilage consists of chondrocytes and extracellular
matrix. The content of cells in cartilage is rare and
amounts to only 1–5% of the cartilage volume w11x. The
main content of extracellular matrix is water. The main
2006/39
 Article in press - uncorrected proof
2 G. Spahn et al.: Water content in hyaline cartilage
substances in the matrix are collagens (approx. 60% of
the dry weight), proteoglycans (approx. 30% of the dry
weight) and proteins as gylcoproteins and non-glycopro-
teins (approx. 10% of the dry weight). Cartilage water is
mostly bound to the aminoglycan side chains of aggre-
can w5, 10x.
Evaluation of water content
Different methods can be used to measure water content
in cartilage specimens. The most simple method is ther-
mogravimetry and determination of the cartilage content
in ash w3x. Mostly the water content in cartilage has been
evaluated by freeze-drying methods. The weight of car-
tilage is measured before and after freeze-drying and the
difference in weight is calculated. The main disadvantage
of this method is the requirement for a relatively large
specimen and the time exposure of several hours w1, 6,
8x.
Many methods for water measurements are based on
chemical reactions (Karl Fischer titration). For example,
organic iodine and sulfuric dioxide react with water,
resulting iodine hydride. This causes a change in the
optical density, which is measurable by photometry:
I2qSO2qH2O™2HIqH2SO4
Phosphorus pentoxide is a very hygroscopic substance
that reacts with water to form ortho-phosphoric acid:
2P2O5q3H2O™2H3PO4
In coulometry, an electrode is laminated with phosphorus
pentoxide. In the presence of water, the phosphorus
pentoxide near the electrode reacts with water. In an
electrochemical reaction, the phosphoric acid is reduced
to hydrogen and oxygen:
4H3PO4™2P2O5q6H2q3O2
The mass of a substance produced at an electrode dur-
ing electrolysis is proportional to the number of moles of
electrons (the quantity of electricity) transferred at that
electrode (Faraday’s law). These measurements are often
used for analysis in chemistry, pharmacology and in
industry w9x.
Hypothesis
Water content determination by a coulometric method
has never been used in the analysis of cartilage com-
position. We hypothesized that this method is able to
determine water content in small cartilage specimens
quickly. It is also hypothesized that water liberated from
the cartilage is a time- and temperature-dependent pro-
cess. The aim of the study was to evaluate differences
between intact and defective cartilage, as well as corre-
lation between the mechanical properties of cartilage and
the water content.
Materials and methods
Specimen and algorithm
A total of 38 fresh sheep knees (17 right and 21 left
knees) from 21 different female sheep obtained at nec-
ropsy were used for the study. The knees were stored at
48C and tests were performed a maximum of 12 h after
death.
Specimens were evaluated using the same procedure.
After arthrotomy, the grade of any cartilage defect was
noted. Exactly 3 min after arthrotomy, the mechanical
hardness was measured. The test for water content
measurement was always started 5 min after opening the
joints.
Grading of cartilage defects
The femoral condyles were prepared and the cartilage
defects within the medial condyles were classified
according to the ICRS protocol w4x. Grading of the car-
tilage defects was undertaken by three independent
investigators (GS, HMK, EK) who are highly experienced
in arthroscopic surgery. The highest grade of cartilage
defects was used for evaluation (Figure 1).
Determination of the mechanical hardness
The mechanical stiffness within the mean weight-bearing
zone of the medial femoral condyle was determined
using an instrument to measure Shore hardness A
(Zwick, Ulm, Germany). Stiffness is a term used to char-
acterize mechanical properties (e.g., in the polymer and
rubber industries). The standard measurement is defined
in DIN 53505. The value is defined as the material resis-
tance against the standardized pressure of a conical
indenter (Figure 2). Shore A is a unit ranging from 0 (com-
plete permeation) to 100 (complete resistance).
Measurement of the water content
After Shore testing, full-thickness cartilage specimens
(approx. 5 mg) were harvested using a biopsy tongue.
Water content measurements were preformed using a
combination of thermogravimetric analysis and coulom-
etry (WDS 400, Sartorius AG, Göttingen, Germany; Figure
3). First the water liberated at room temperature was reg-
istered. All specimens liberated water from the surface
rapidly up to a first peak (Figure 4). After reaching this
peak, the specimens were subjected to continuous heat-
ing up to 1058C. The system was calibrated using sodi-
um tungstate, which has a highly constant water content
of 1.07%.
Statistics
Statistical analyses were performed on a personal com-
puter using SPSS v. 13.0 (SPSS Inc., Chicago, IL, USA).
After testing for normal distribution and variance homo-
geneity, one-way analysis of variance (ANOVA) and post
hoc pairwise comparison of the means were preformed.
Pearson correlation coefficients were used to examine
the relationships between the parameters. A p-value
-0.05 was considered significant.
 Article in press - uncorrected proof
Figure 1 Grading of chondral defects (ICRS protocol) w4x.
(A) Grading system for chondral lesions according to the ICRS
protocol. (B) Grade I lesion of the medial femoral condyle (soft-
ening and superficial fissuring).
Results
Frequency of cartilage defects
In 20 knees the cartilage was intact. In the remaining
knees, 14 had a grade I defect and 4 a grade II defect.
Water content
Water liberation from the surface of intact cartilage spec-
imens was significantly slower than from cartilage spec-
imens with defects (Table 1).
G. Spahn et al.: Water content in hyaline cartilage 3
Figure 2 Measurement of cartilage stiffness by the Shore A
test.
Mechanical hardness is a property of solid material reflecting its
resistance to permanent deformation, and correlates with the
stiffness (DIN 53505). (A) The top of a steel cone (0.79 mm, angle
358) is intended to the surface with a constant power of 50 pro-
duced by a precision spring. (B) During experiments, the device
was placed on the cartilage surface within the mean weight-
bearing zone of the medial femoral condyle for exactly 10 s.
Cartilage specimen with defects had significantly high-
er superficial water content than specimens from intact
cartilage (Table 1). The time for water liberation from sur-
faces tended to be shorter for grade II defects (p-0.236)
than for grade I defects. This correlated with the tenden-
cy for higher superficial water content in grade II defects
 Article in press - uncorrected proof
4 G. Spahn et al.: Water content in hyaline cartilage
Figure 3 Principle of water content measurement.
(With generous permission from Sartorius AG, Göttingen,
Germany.)
Figure 4 Measurement of water content.
In the first step, water liberated from the specimens at room
temperature (water from the specimen surface) was registered.
The time and percentage water content at this time (peak) were
measured. In the next step, specimens were subjected to con-
tinuous heating up to 1058C. The water content was measured
after 15 min. The integral of the area correlates with the water
content.
Table 1 ((•••author: give heading, please•••)).
Cartilage defect
Intact (ns20)
Evaporation time for superficial water (s) 3.9"0.5
Superficial water content (%) 3.6"1.0
Water content (%) 66.9"4.8
Mechanical hardness (Shore A) 90.5"5.6
compared with grade I defects (p-0.270). The water
content in grade II defects was significantly higher
(p-0.008) than in grade I defects.
Mechanical hardness
Chondral damage correlated significantly with lower
mechanical hardness. Significantly lower Shore hardness
was measured for cartilage defects compared to intact
cartilage (Table 1). No significant differences were found
between grade I and II defects (p-0.752).
Correlation between mechanical hardness and
water content
The mechanical hardness correlated significantly
(Rs0.540, p-0.000) with water content (Figure 5). Cor-
relation between mechanical hardness and superficial
water liberation (Rs0.495, p-0.02), as well as time of
liberation (Rs0.757, p-0.000), was also significant (Fig-
ure 6).
Discussion
Water is the most abundant component (60–80% of the
net weight) of hyaline cartilage. Proteoglycans together
with collagen molecules generate a network with a very
high water-binding capacity. Water is mostly bound to
the side chains of aggrecan molecules. The biochemical
composition of the extracellular matrix confers the vis-
coelasticity and mechanical resistance of healthy carti-
lage.
Edema, swelling and softening are characteristic for
the early stages of cartilage degeneration. This patholog-
ical process is accompanied with an increase in water
content w2, 7, 12x.
In our experiment we found an approximately 7% high-
er water content in cartilage defects. The experimental
design of our measurement allowed determination of the
water content in very small cartilage specimens within a
short time interval of 15 min. The accuracy of the devices
for water quantification is 1 mg.
During the experiment, water liberation by evaporation
of approximately 3–5% within 170–230 s was observed
at room temperature. This water liberation conforms to
water that adheres to the surface of the specimen w9x.
This ‘‘superficial water content’’ was significantly higher
in cartilage defects. The liberation of superficial water
required a significantly shorter time interval in the case
of cartilage defects than for intact cartilage.
The reason for this observation is still unclear. First,
cartilage defects may contain more free water (non-cova-
p
Grade I (ns14) Grade II (ns4)
2.5"0.4 2.8"0.2 -0.000
4.9"1.6 5.9"0.6 -0.001
72.6"3.1 77.8"2.7 -0.000
64.1"6.5 65.2"6.2 -0.000
 Article in press - uncorrected proof
Figure 5 Correlation between water content and mechanical
hardness.
The mechanical hardness is significantly correlated (Rs0.540,
p-0.000) with the water content.
lently bound) that is more quickly liberated by simple
evaporation. Second, it is also possible that the de-
creased mechanical resistance of defective cartilage
plays a role. The specimens were obtained using an
arthroscopic tongue, which squeezes the cartilage. This
mechanical alteration causes water liberation. Third, car-
tilage from degenerated surfaces may have a more fluid
film on their surfaces.
We found a significant correlation between mechanical
hardness and the water content. The softening of carti-
lage in low-grade degeneration is likely produced by
chondral edema, in which ‘‘superficial water’’ possibly
plays an important role.
Cartilage is a very susceptible and unstable tissue. At
room temperature, cartilage specimens lose approxi-
Figure 6 Correlation of surface water content and mechanical
hardness.
The mechanical hardness is significantly correlated (Rs0.384,
p-0.017) with the surface water content.
View publication stats
G. Spahn et al.: Water content in hyaline cartilage 5
mately 3–5% of their net weight by simple evaporation.
This must be considered in future experiments.
Conclusion
Coulometry, e.g., by Karl Fischer titration or using a WDS
400 instrument, is a very sensitive method for evaluation
of water content in cartilage. The accuracy of the tests
is approximately 1 mg of water. The main advantage is
the short time required for measurements. Small speci-
mens of approximately 5 mg are sufficient for an exact
analysis. Water liberation from cartilage is a temperature-
dependent process. Up to 5% of the total water is lib-
erated by simple evaporation within a few minutes. The
higher water content and spontaneous evaporation in
cartilage defects correlate with lower mechanical hard-
ness.
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