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CHARACTERISATION OF CASSAVA FIBRE FOR USE AS A BIOMATERIAL

Article  in  International Journal of Engineering Science and Technology · July 2012

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 Lois Larbie et al. / International Journal of Engineering Science and Technology (IJEST)

CHARACTERISATION OF CASSAVA
FIBRE FOR USE AS A BIOMATERIAL
Lois Larbie, Claude Fiifi Hayford, Elsie Effah Kaufmann (PhD)*
Department of Biomedical Engineering, University of Ghana, Legon
Accra, Ghana
eek@ug.edu.gh

Abstract:
In this study we investigate the cytotoxicity of de-starched cassava fibre granules and fine powder using human
peripheral blood mononuclear cells (PBMC) and examine changes in the composition of Simulated Body Fluid
(SBF) resulting from immersion of cassava fibre samples. The purpose of the study was to characterise cassava
fibre for possible biomaterial applications. Preliminary results indicate insignificant cytotoxic effects on PBMCs
with cassava sample concentrations of 0.1g/ml, 0.025g/ml and 0.00625g/ml. Additionally there was little or no
significant change in Na, K, Mg, Cl, Ca, Mn, and Cu concentrations upon immersion in SBF as observed over a
one week period at a temperature of 37°C. These initial results suggest cassava fibre may be considered for
biomaterial applications following more extensive characterisation.

Keywords: cassava fibre; biomaterial; cytotoxicity; immersion characteristics; Instrumental Neutron Activation
Analysis (INAA)

1. Introduction
Cassava known biologically as Manihot esculenta Crantz is one of the major and popular foods consumed in
Ghana [Ennin et al., (2009); IFAD, (2005)]. It is used in the preparation of various kinds of food in the country.
The peel also serves as food for livestock such as sheep and goats. Starch extracted from cassava has found uses
in various industries and the fibre is being used in biodegradable plastics [Sriroth and Sangseethong, (2006)], for
disposables used in packaging and in homes.

As a national commodity, cassava production contributes about 22% to Ghana’s agricultural gross domestic
product (AGDP) [Ennin et al., (2009)]. Presently, large scale “small-holders” commercial farming of cassava is
being encouraged to feed the US$ 7 million Ayensu Starch Factory under the President’s Special Initiative
Scheme for rural employment and wealth creation [Addy et al., (2004)]. The industrial use of cassava, however,
leaves in its wake tonnes of waste to be disposed. This residue could accrue some economic benefit to the
country if some use could be found for it.

In the field of Biomedical Engineering, cassava fibre has yet to find any meaningful application. However,
recent interest has seen cassava starch and flour finding widespread use in the development of biodegradable
polymer blends and bio-composites [Kim et al., (2011)] for tissue scaffolds [Sunthornvarabhas et al., (2011)]
showing advantageous properties such as good biocompatibility, good mechanical properties and non-toxic
degradation products [Lu et al., 2009)].

This study forms part of a larger study aimed at reducing waste by finding use for the cassava fibre by-product
from the starch extraction process by studying the cytotoxicity using a lactate dehydrogenase (LDH) test as a
measure and also determining the biodegradability characteristics of the de-starched cassava fibre for potential
use as a biomaterial.

Cytotoxicity testing of biomaterials is targeted mainly at substances which leach out into surrounding tissue
[Idris et al., (2010)] that could have potentially adverse effects on these tissues. The aim of in vitro (cell
culturing) cytotoxicity tests is to detect the potential ability of a device or material to induce sub-lethal or lethal
effects as observed at the cellular level. According to ISO 10993-1, the in vitro cytotoxicity assay is one of two
tests that must be considered in the evaluation of all biomedical device categories; the other test being the
sensitization test [Wallin and Arscott, (1998)].

Lactate dehydrogenase (LDH) assays are useful in determining the cytotoxic potential of compounds [Dubar et
al., (1993); Kondo et al., (1993); Murphy et al., (1993); Courjault et al., (1993); Shrivastava et al., (1992);

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Gelderblom et al., (1993); Thomas et al., (1993); Sasaki et al., (1992)].  LDH is a stable cytoplasmic enzyme
which is present in most cells. It is released into the cell culture supernatant upon damage of the cytoplasmic
membrane [Korzeniewski and Callewaert, (1983)]. The LDH activity is determined in an enzymatic test. The
first step is the reduction of NAD+ to NADH/H+ by the LDH catalysed conversion of lactate to pyruvate. In a
second step, the catalyst (diaphorase) transfers H/H+ from NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-
(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), which is reduced to a red formazan [Decker and Lohmann-
Matthes, (1988); Lappalainen et al., (1994); Nachlas et al., (1960)]. An increase in the number of dead or plasma
membrane-damaged cells leads to an increase of the LDH enzyme activity in the culture supernatant. This
allows the LDH test to be used as a general marker of injury to cells (Takara Kit Manual, Japan). A negative test
result indicates that a material is free of harmful extractables or has an insufficient quantity of them to cause
acute effects under exaggerated conditions with isolated cells. A positive result can be taken as an early
warning sign that a material contains one or more extractable substances that could be of clinical importance and
therefore requires further investigation to determine the utility of the material in a given application [Wallin and
Arscott, (1998)] and as a medical device like an implant.

Investigations in cell-free solutions with ionic concentrations similar to body fluid allow the study of the
chemical and mineralogical changes of implants under conditions that simulate the physiological interactions
between the material surface and the implant site [Kokubo et al., (1990); Ratner et al., (1996)]. Cassava
generally has mineral content similar to blood [Rojas et al., (2007)] and these could possibly have an impact on
the physiological environment as the body tries to maintain a fine balance of ion concentration. It is therefore
imperative to know the contribution of ions cassava fibre would make in the physiological environment and
from this deduce whether these are in toxic amounts.

2. Materials and Methods

2.1. Cassava fibre preparation

Two varieties of cassava tubers were used: the IFAD variety from the Biotechnology and Nuclear Agricultural
Research Institute (BNARI), Ghana and the Akilakpa Middle variety from the Department of Botany, University
of Ghana. The cassava tubers were washed, peeled and washed again. Using a normal kitchen grater, the tubers
were shredded to produce small fibre sizes. The 6 mm diameter section of the grater was used for the fibre for
the leaching/immersion study. The shredded product was washed several times to remove as much of the starch
content as possible. The shredded product was also soaked to aid in achieving low starch content. This was to
help obtain a product that had similar properties as that from starch extraction at the processing factory. A
muslin cloth was used to squeeze the sample after washing and dried under room temperature. The dried sample
from the different varieties was retrieved and packaged separately for future use. Some of the fibre obtained
from the Akilakpa Middle variety was processed into fine powder using an electronic mill.  The average particle
size was microscopically determined using an eyepiece graticule and a stage micrometer. The granules had an
average particle size of 38 µm while the powder particle size was 19 µm.

2.2. Sample preparation for immersion study

Simulated Body Fluid (SBF) was prepared by weighing out 5.096 g of Trizma Base (SIGMA, Lot 89H5436),
7.30 g of NaCl (Scientific and Chemical Supplies Ltd., Batch# 50519), 0.234 g of KCl (SIGMA, Batch#
085K0061), 2.368 g of NaHCO3 (Merck, Lot 6329.1000), 0.123 g of MgCl2.6H2O (SIGMA, Lot 98H0262 ),
0.099 g of MgSO4 (anhydrous), 0.174 g of K2HPO4 (Merck) and 0.276 g of CaCl2 (anhydrous) (Fluka Chemie
AG). These were added to 500 ml of distilled deionised water in the order stated, one after the complete
dissolution of the other. The solution was prepared using a magnetic stirrer. The pH of the prepared solution was
measured to be 9.07. This was brought up to 7.57 at 27.4°C and made up to 1L by adding distilled deionised
water. The pH meter was calibrated with 4.01 and 7.01 pH solutions before use. Under the bio-safety hood, the
SBF was filter sterilised and stored at 4°C for later use.

As part of the sample preparation, the packaged dry shredded cassava was retrieved, and 0.5g weighed into petri
dishes. 1.5ml of 70% ethanol was then pipetted over them to sterilize them. This was done under a bio-safety
hood. The dry sterilised samples were then transferred into fifteen 15 ml sterile tubes and covered.

The immersion of the fibres involved pipetting 10 ml of the SBF unto the samples in the 15 tubes. This was to
achieve an average mass-to-volume ratio of 0.050 g/ml. The same volume of SBF was again pipetted into
another set of 15 sterile tubes. Two sterile tubes were loaded with 0.5 g each of the sterilised cassava fibre. The
32 sample tubes were loaded in a test tube rack and placed in an incubator at 37°C. The samples with fibre and
SBF were occasionally rocked. At the specified endpoints of 1 day, 4 days and 7 days after immersion, the

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supernatant was decanted off three samples into new sterile tubes. Three SBF only samples were also retrieved
at each endpoint. These were stored in a refrigerator at 4°C for later analysis. The immersion and decantation
procedures were all carried out under the bio-safety hood.

Instrumental Neutron Activation Analysis (INAA) was then employed for the determination of elemental
concentration in the end-point solutions.

2.3. Sample irradiation

End-point solutions were shaken in their containers to ensure uniformity before weighing. 500 µL of liquid
sample were weighed in triplicate for each test material into a clean polyethylene capsule and heat sealed with
soldering rod. 200 mg of cassava fibre sample was also weighed and prepared as indicated. Each sample was in
turn put into a rabbit capsule and smoothly heat sealed with a soldering rod. Single standard elements of
concentration 10 mg/L and 20 mg/L of the elements of interest were also prepared in the same manner as the
test samples. The polyethylene capsule of diameter 1.2 cm and height 2.3 cm containing the liquid samples were
then put into bigger polyethylene capsules of diameter 1.6 cm and height 5.5 cm (Rabbit capsule), that is double
encapsulation. Two small capsules were also prepared with the 70% ethanol used for sterilization as a control.

Samples and controls were irradiated in the Ghana Research Reactor (GHARR-1) at the Ghana Atomic Energy
Commission, operating at 15 KW at a thermal flux of 5×1011 ncm-2s-1. Samples were transferred into irradiation
sites via a pneumatic transfer system at a pressure of 0.6 MPa. The irradiation was categorized according to the
half-life of the element of interest. For 27Mg, 38Cl, 49Ca, 56Mn, and 66Cu samples irradiation was done for two
minutes and radiation was counted for ten minutes. For medium-lived radionuclides like 24Na, and 42K both end-
point solution samples and cassava fibre samples were irradiated for one hour and delayed for 24hrs with 10
minutes counting. After the irradiation, radioactivity measurement of induced radionuclide was performed by a
PC-based γ-ray spectrometry set-up. It consists of an n-type High Purity Germanium (HPGe) detector coupled
to a computer based multi-channel analyser (MCA) via electronic modules. The relative efficiency of detector
is 25% and its energy resolution of 1.8 keV at a γ-ray energy of 1332 keV belonging to 60Co. Through
appropriate choice of cooling-time, detector’s dead time was controlled to be less than 10%. Identification of γ-
ray of product radionuclide was identified through the energies and quantitative analysis of the concentration
was achieved using the γ-ray spectrum analysis software, ORTEC MAESTRO-32.

The results obtained from the analysis were statistically evaluated with a statistical tool, SPSS 16.0, to determine
the differences in means of the concentrations of the selected elements at the various endpoints. The analysis
was performed at the 95% confidence interval and the 0.05 significance level. For the analysis all values less
than 0.01 mg/l were assumed to be 0.01 mg/l.

2.4. Cytotoxicity testing

Using a butterfly needle, blood was drawn from a healthy donor into an EDTA vacutainer tube. The blood was
then diluted twice using RO (made by adding 500 ml of RPMI 1640 (Lot: 46k2323 Cat No: 125886) unto 5 ml
of L-glutamine (GIBCO Powder Lot: 9825) and 5 ml penstrep (SIGMA Lot: 66K2319) after which 45 ml of the
resulting solution was pipetted into accuspin tubes and spun at 2000 rpm for 10 minutes. The supernatant was
pipetted off leaving the mononuclear cell suspension (MCS) which was transferred into a 50ml centrifuge tube.
R5 (made of RO and 5% by volume of Foetal Bovine Serum (FBS)) was added to the MCS to make it up to 50
ml. This was centrifuged at 2000rpm for 10 minutes. The supernatant was pipetted off and the mononuclear
cells washed again using R5 and the same process.

The mononuclear pellet obtained after washing was then suspended in 3-5 ml of HR10 (500 ml of RPMI 1640, 5
ml of L-glutamine, 5 ml of penstrep and 10% FBS) after which 25 µl of the cell suspension was diluted using
HR10. For counting, 20 µl of the diluted cell suspension was mixed with 20 µl of Trypan Blue and the cells
counted using a Haemocytometer under 40X magnification of a light microscope. Cell concentration was
estimated and then adjusted to 2.0 x 106 cells/ml. Unused cells were cryopreserved.

Cell plating was carried out by adding 100 µl/well of 2.0 x 106 cells/ml to the plate. The antigen/solution was
added at the desired concentration along with the cassava fibre sample to the wells as per the template. Three
controls were used. For the negative control, the wells contained only the cells and the culture medium with no
cassava fibre sample. The positive control had similar constitution but with the cells lysed completely for total
LDH analysis. To cancel out any anomalies from background readings arising from alcohol traces in the sample,
the alcohol used for sterilization was also used as a control. To this effect, 5 µl of alcohol was added to wells
containing only cells and culture. The plate was sprayed with alcohol and incubated in a carbon dioxide
incubator set at 37 °C for 24 hours.

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The supernatant was harvested (without picking up cells) into appropriately labelled Eppendorf tubes and stored
in a freezer for later use. The negative and alcohol controls were harvested just like the assay by only picking up
the supernatant. For the positive control, the complete contents of positive control labelled wells was picked up
and lysed. If lysing was not done immediately, samples were stored in cryo tubes in the freezer. Lysing was
achieved by the Freeze/Thaw technique using liquid nitrogen and a water bath. The lysed cell samples were
spun for 20 minutes at 10 rpm to separate the supernatant from the damaged cells. The supernatant was drawn
out into labelled Eppendorf tubes for freezing with the rest of the samples for later use.

LDH was measured by transferring supernatants into corresponding wells of a 96-well plate and 100 µl of
reaction mixture (Takara Cytotoxicity LDH Measurement Kit) added to each well. This was then incubated for
30 minutes in the dark (protected from light) after which absorbance at a wavelength of 490-492 mm was
measured using a plate reader. The relative LDH concentration was calculated using the formula:

Cytotoxicity was then determined using statistical tools to evaluate if the various relative LDH concentrations of
the assays were statistically different from that of the positive control.

3. Results and Discussion

3.1. Immersion study

Statistical analysis of results obtained from the immersion of the cassava fibre in SBF showed no significant
differences between elemental concentrations for samples from the three endpoints. The trend was the same for
SBF only samples with the exception of results for Cl which showed a significant difference in concentration at
3 days compared to 4 and 7 days of treatment. Similarly comparison of mean concentrations from samples with
cassava fibre and SBF only samples showed no significant difference at the same endpoint for each element of
interest. Table 1 shows results obtained after using the γ-ray spectrum analysis software.
Table 1. Concentration of elements in endpoint solutions from γ-ray spectrum analysis software

Endpoint Sample Sample Concentrations (mg/l)

no. source id Mg Cu Cl Mn Ca Na K
A 4.87±0.73 0.02 2576±386 4.68±0.70 1.06±0.16 377±56.55 23.31±3.50
I B 7.16±1.07 <0.01 2420±363 3.27±0.49 1.05±0.16 152.60±22.89 6.92±1.04
C 4.62±0.69 0.10±0.02 1664±250 11.75±1.76 5.85±0.88 430.26±64.54 14.76±2.21
1
D 0.44±0.07 <0.01 27849±4177 <0.01 5.17±0.78 398.35±59.75 6.36±0.95
II E 1.42±0.21 <0.01 2288±343 <0.01 1.32±0.20 26.09±3.91 11.72±1.76
F 13.22±1.98 0.03 2258±339 5.13±0.77 2.99±0.45 159.31±23.90 6.45±0.97
G <0.01 <0.01 23586±3538 2.55±0.38 <0.01 420.45±63.07 2.35±0.35
I H 3.17±0.48 <0.01 2323±348 0.73±0.11 3.44±0.52 306.49±45.97 7.47±1.12
I 4.10±0.62 0.12±0.02 1431±215 2.87±0.43 6.25±0.94 340.77±51.12 8.79±1.32
2
J 2.45±0.27 0.11±0.02 2287±343 3.39±0.51 3.42±0.51 422.53±63.38 8.93±1.34
II K 2.86±0.43 0.10±0.02 2383±348 <0.01 10.37±1.56 262.57±39.39 6.85±1.03
L 8.11±1.22 0.06±0.01 2233±335 4.85±0.73 6.13±0.92 357.14±53.57 12.90±1.94
M 2.29±0.34 <0.01 2217±333 1.45±0.22 8.68±1.30 244.87±36.73 2.15±0.32
I N 2.81±0.42 0.02 1638±246 2.89±0.43 3.54±0.53 316.48±47.47 5.59±0.84
O 9.92±1.45 0.05±0.01 2280±342 2.06±0.31 12.23±1.83 294.01±44.10 10.39±1.56
3
P 5.69±0.85 <0.01 2094±314 20.7±3.105 <0.01 188.43±28.26 7.09±1.06
II Q 2.83±0.42 0.29±0.04 2269±314 1.46±0.22 6.80±1.02 245.01±36.75 10.88±1.63
R 0.09±0.01 0.12±0.02 2086±313 <0.01 8.52±1.28 372.93±55.94 9.58±1.44
70%
T 0.55±0.08 0.32±0.05 182.58±27.40 2.82±0.42 3.41±0.51 22.95±3.44 4.79±0.72
Alc.

I-immersed fibre solution II-SBF only solution. For endpoint 1(3 days immersion), 2 (4 days immersion) and 3 (7 days immersion). 70%
Alc. was the negative control.

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The limit of detection for the INAA was 0.01 mg/l and values less than this were approximated to 0.0l mg/l. For
the various endpoints, there was found to be no significant differences in means for the concentrations of the
elements analysed (both SBF only and immersion samples). This could be an indication that there was no
leaching of these elements from the cassava fibre into the simulated body fluid. There was also no significant
difference in means for the concentrations of the elements in the control samples (SBF only) for the various
endpoints. This was generally expected as there was no cassava fibre present to leach mineral into the solution.
From the raw data, Cu and Mn which in this study were regarded as trace elements, in several samples fell
below the detection limit of 0.01 mg/l.

Elemental Conc. Comparison at 3 Different Endpoints

1.0×10 5

Treatment Period

* 3 days
1.0×10 4 4 days
7 days

1.0×10 3
Log [Mean Concentration](mg/L)

1.0×10 2

1.0×10 1

1.0×10 0

1.0×10 -1

1.0×10 -2
s)

i)

s)

i)

(i)

s)

i)

s)

i)

s)

i)
l(s

l(i

(s
a(

u(

g(

n(

a(
u(

g(

n(
a(

a(
K
C

K
C

N
C

M
C

N
C

Elements Analyse d
*
significant difference in concentration at 3 days compared to 4 and 7 days for Cl(s)
 
Figure 1-Elemental concentrations in SBF(s) only and immersed (i) samples over a period of one week

Using a paired sample t-test with a confidence interval (CI) of 95%, the means of elemental concentrations in
both immersion samples and SBF-only samples at each endpoint were analysed to determine if the results
showed any differences. Again, results showed no significant difference in means for each element considered
with the exception of that for Mn which showed a significant difference between concentration in immersion
samples and that for SBF at the first endpoint of three days.

Comparison of the raw means for SBF only and endpoint solutions for the various elements at each of the three
endpoints showed no clear trend. In some cases, the values for the SBF only solution were higher than those of
the endpoint solutions for the same endpoint whereas in others the means obtained for the endpoint solutions
decreased from endpoint 1 to endpoint 3 with others showing peaks in between. The general expectation was
that if the elements were going to leach out into the solution, there would be an increase in original
concentration in the SBF solution as immersion time increased. Results of possible ion deposition from SBF
onto cassava fibre samples will be presented in a future study. The figures below show the mean concentrations
of the various elements of interest in immersed samples as compared to those in SBF only samples over the
period of study.

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Comparison of [Ca] in endpoint solutions

1
1.5×10
Ca(s)

Mean Concentration(mg/l)
Ca(i)
1
1.0×10

5.0×10 0

0
3 4 7
Period of Treatment (days)

Figure 2-Comparison of mean [Ca] in immersed fibre (i) and SBF (s) solutions over 1 week

Comparison of [Cl] in endpoint solutions

2.5×10 4
Mean Concentration(mg/l)

2.0×10 4 Cl(s)
Cl(i)
1.5×10 4

1.0×10 4

5.0×10 3

0
3 4 7
Period of Treatment (days)
 
Figure 3-Comparison of mean [Cl] in immersed fibre (i) and SBF (s) solutions over 1 week

Comparison of [Na] in endpoint solutions

5.0×10 2
Mean Concentration(mg/l)

4.0×10 2 Na(s)
Na(i)
3.0×10 2

2.0×10 2

1.0×10 2

0
3 4 7
Period of Treatment (days)
 
Figure 4-Comparison of mean [Na] in immersed fibre (i) and SBF (s) solutions over 1 week

Comparison of [Mn] in endpoint solutions

1.5×10 1
Mean Concentration(mg/l)

Mn(s)
Mn(i)
1.0×10 1

*
5.0×10 0

0
3 4 7
Period of Treatment (days)
*
significant difference between Ca(s) and Ca(i)
 
Figure 5-Comparison of mean [Mn] in immersed fibre (i) and SBF (s) solutions over 1 week

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Comparison of [Mg] in endpoint solutions

1.0×10 1

Mean Concentration(mg/l)
8.0×10 0 Mg(s)
Mg(i)
6.0×10 0

4.0×10 0

2.0×10 0

0
3 4 7
Period of Treatment (days)
 
Figure 6-Comparison of mean [Mg] in immersed fibre (i) and SBF (s) solutions over 1 week

Comparison of [Cu] in endpoint solutions

2.5×10 -1
Mean Concentration(mg/l)

2.0×10 -1 Cu(s)
Cu(i)
1.5×10 -1

1.0×10 -1

5.0×10 -2

0
3 4 7
Period of Treatment (days)
 
Figure 7-Comparison of mean [Cu] in immersed fibre (i) and SBF (s) solutions over 1 week

Comparison of [K] in endpoint solutions

2.5×10 1
Mean Concentration(mg/l)

2.0×10 1 K(s)
K(i)
1.5×10 1

1.0×10 1

5.0×10 0

0
3 4 7
Period of Treatment (days)
 
Figure 8-Comparison of mean [K] in immersed fibre (i) and SBF (s) solutions over 1 week

Some elements analysed showed decreases in concentration for some endpoints. This is interesting because it is
possible that these elements have been picked up by the immersed fibre samples and if that is the case,
important implications arise. One such implication is that the fibre has the potential of picking up ions from
solution thereby causing a change in physiological fluid ion balance. Additionally, modifications in implant
surface characteristics in the presence of physiological solutions have been suggested to affect biological
response [Roach et al., (2007)]. The degree of adsorption of these elements would have to be investigated by
analysing immersed fibre samples to ascertain that these elements were actually picked up by the fibre.

3.2. Cytotoxicity testing

From the Beer-Lambert theory the absorbance of a sample depends upon its overall thickness and the
concentration of the light absorbing species [Hallet et al., (1992)]. Thus an increase in absorbance means a
relative increase of the Lactate Dehydrogenase enzyme concentration in the medium. The LDH enzyme is
released when the cell membrane is damaged due to exposure to a toxic component [Takara Kit Manual, Japan].
A damaged cell membrane results in cell death, and so more cells die when there are highly toxic or large
quantities of toxic components leading to a corresponding rise in the concentration of LDH enzyme released.

The essence of the positive control was to identify the maximum amount of LDH that can be released from the
cells. Thus a material that induces the discharge of an equivalent amount of LDH as in the positive control can
be said to be cytotoxic. To determine whether the cassava sample was cytotoxic or not, the various relative LDH

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concentrations measured for the fibres were evaluated for statistical difference from that of the positive control
(positive controls - lysed cells, negative control- no fibre sample and alcohol control).

Levels of LDH Expressed

1.5
Powder
Granules

Relative [LDH]
1.0

0.5

0.0
+ve Ctrl 0.1 0.025 0.00625
Sample Concentration (g/ml)
 
Figure 9-Relative LDH Concentration Expressed for Different Sample Concentrations in PBMC culture. Negative control values used as
background and subtracted from all.

The cassava fibre was identified as cytotoxic when the relative [LDH] for the various concentrations was above
a specified value of 20%, i.e. an equivalence of 0.2 from the graph in Fig.9. From the results above, it was
observed that except for the 0.1 g/ml, values for both granules and powder samples were low and fell within the
non-cytotoxic range, (they were actually 10% or below). This means that the lactate dehydrogenase being
released due to cell membrane damage of the Peripheral Blood Mononuclear Cells caused by the presence of the
investigated concentrations of cassava fibre is not sufficient to cause adverse effects to the host cell.

From literature there are other causes of cell death not directly related to the passive presence of a material
[Zong and Thompson, (2006)]. Cell death can be caused by the mobility of cells. The relative movement of the
cultured cells can cause the crushing of the cells against each other leading to damage of the cell membrane.
Cell death may also be caused by apoptosis. Cell death can even be caused by the nutrient medium used during
cell culturing. In as much as di-molecular oxygen is an essential ingredient of cell culture systems, oxygen is
also a potent mediator of toxicity in cell culture. The essence of the negative control was therefore to cancel out
or neutralize any anomalies that may have arisen due to cell membrane damage caused by the immediate factors
listed above. Thus LDH concentrations measured for the negative control was considered as background and
subtracted from the assay results as well as the positive control.

The results obtained indicate that the form of sample used (powder or granules) do not significantly alter the
outcome of the cytotoxicity test. Independent two-tailed t-tests at 5% level of confidence showed no significant
statistical differences among the different concentrations of fibre.

Conclusion
The preliminary results obtained indicate that the cassava fibre used is not cytotoxic at the concentrations
investigated. The finding is supported by the results from the immersion studies that indicate that the immersed
fibre samples do not release ions into the simulated fluid but might rather influence physiologic fluid
composition by picking up ions from solution instead, a fact that must be ascertained by further tests. It is
recommended that other sterilization techniques such as gas plasma and filtration for elution be tried and the
immersion studies expanded to cover the detection of any macromolecules that may be present due to
degradation of the fibre.

Acknowledgements
The authors acknowledge the support of Mr. Achel, Dr. Asare, Professor I.K. Asante, Dr. Daniel Ofori, the
Ghana Atomic Energy Commission and the Noguchi Memorial Institute for Medical Research in making
available their facilities and resources for this research and for their continued support of this work.

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 Lois Larbie et al. / International Journal of Engineering Science and Technology (IJEST)

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