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Characterization and Development of Optimization Strategy for the Processing of
Allogenic and Xenogenic Bone and Pericardium
Submitted to
The Faculty of Engineering at the Friedrich-Alexander University of
Erlangen-Nuremberg
to obtain the degree
DOKTOR-INGENIEUR
presented by
Mohannad Qasim Mustafa Marashdeh
Erlangen 2007
As dissertation approved by The Faculty of Engineering Science of the Friedrich-Alexander University of Erlangen-Nuremberg
Day of submission: 17.04.2007
Day of examination: 06.06.2007
Dean: Prof. Dr.-Ing. A. Leipertz
Examiners: Prof. Dr. R. Buchholz
Prof. Dr. P. Greil
Charakterisierung und Entwicklung einer Optimierungsstrategie für die Prozessierung von allogenen und xenogenen Knochen und Perikard
Der Technischen Fakultät
der Friedrich-Alexander Universität Erlangen-Nürnberg
zur Erlangung des Grades
DOKTOR-INGENIEUR
vorgelegt von
Mohannad Qasim Mustafa Marashdeh
Erlangen 2007
Als Dissertation genehmigt von der Technischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der Einreichung: 17.04.2007
Tag der Promotion: 06.06.2007
Dekan: Prof. Dr.-Ing. A. Leipertz
Berichterstatter: Prof. Dr. R. Buchholz
Prof. Dr. P. Greil
Acknowledgment
Acknowledgment I would like to express my gratitude to all those who gave me the possibility to complete this thesis. First of all, I want to thank Prof. Rainer Buchholz and Prof. Thomas Neeße for their constant guidance, support and encouragement. I am deeply indebted to my supervisor Dr. Roman Breiter whose motivations and stimulating suggestions helped me during the research and writing phases of this thesis. I would like to thank the Examination board (Prof. Greil and Prof. Pischetsrieder) for their valuable criticism and evaluating the present work. Special thanks to the company Tutogen Medical GmbH for the financial support during this thesis. My sincere thanks go to Dr. Dueck and Dr. Georgiadis for their valuable advices and support during the work. I’m also grateful for Silke Schwarz, Ludwig Körber and Ana Herakovic for the helpful collaboration during the research phase. I would like to thank the staff of LUR and BVT Erlangen as well as of Tutogen Medical GmbH for their help and support. I am deeply indebted to those who have participated in reviewing the present work, especially Khaled Abderrzaq and his wife Rana Al-Rabei. Finally, I would like to express my deepest, warmest and endless gratitude to my parents, brothers and sisters for their patience, enthusiastically supporting and unlimited encouragement
Abstract
I
Abstract Allografts and xenografts are used as alternatives to autografts, however the concern
about the immunological reaction and the transmission of host diseases are the main
limitations coupled with the use of these grafts. Therefore these grafts have to be
preprocessed before being used. Unfortunately, the preprocessing treatments could
destruct the biological and structural integrity of the tissues. Tutoplast® process is a
comprehensive process for the conservation of the allo- and xenografts.
During this work, the influence of Tutoplast process on the stability of collagenous
tissues was examined. For this purpose, measurements of the fraction of denatured
collagen (DC), measurements of isotonic shrinkage temperature, SDS-PAGE
investigations and the mechanical properties were used to evaluate the quality of the
tissues.
It was proved that the processing induces certain structural destruction or worsening of
the quality of tissues. Therefore, it was reasonable to follow the contribution of each step
in the process in this destruction.
It was found that the 1 N NaOH treatment in the process induces amino acids
modification yielding tissues with lower thermal stability, however this could be
reversible. It was observed that treating the tissues with 1 N CH3COOH is not suitable to
restore the tissues to their physiological state. The best variant was to treat the tissues
with 0.1 N CH3COOH followed by 1-2 10-min water baths.
The 3 % H2O2 had little effect on the quality of the tissues. Furthermore, 10 % H2O2
could be used to guarantee the oxidation of soluble proteins and the inactivation of
viruses without almost further worsening of the quality of tissues.
The pure acetone treatment used in the process was found to be more effective than the
graded acetone treatment as dehydrating agent; however the 2-week treatment is too long.
It was observed that the tissues were fully dehydrated after 2 days. Further treatment with
acetone leads to avoidable volume shrinkage of the tissues.
The structural heterogeneity and the fiber orientation were dominant during the
characterization of the mechanical properties, which made the analysis of the results
complicated.
Summary
II
Summary The autograft is considered as the gold-standard graft in the medical field because it
contains viable cells and growth factors, which stimulates the healing of the graft.
However, the limited availability and the additional morbidity are the main disadvantages
associated with the transplantation of autografts. Therefore, alternative grafting materials
have been always used to fulfill the increasing demand for grafts in the medical field.
Allografts and xenografts are used as alternatives to autografts, however the concern
about the immunological reaction and the transmission of host diseases are the main
limitations coupled with the use of these grafts. Therefore these grafts have to be
preprocessed before being used. Unfortunately, the preprocessing treatments could
destruct the biological and structural integrity of the tissues.
The present work aims to examine the possibilities to optimize a process for the
conservation and processing of bone and soft tissue allo- and xenografts (the Tutoplast®
process). In order to perform an optimization of the process, first the influence or the
modifications induced by the process were defined carefully. Second, the effect of each
step of the process on the stability of the tissues was studied separately. Finally, time-
concentration modifications or alternative steps were evaluated.
During this work, measurements of the fraction of denatured collagen (DC) after
selective enzymatic digestion technique, measurements of isotonic shrinkage
temperature, SDS-PAGE investigations and the mechanical properties were used to
evaluate the quality of the tissues.
The bovine bones, used in this work, were first pulverized under liquid nitrogen to
accelerate the demineralization of the bones, which is necessary before the enzymatic
digestion of bone samples. The bone powder from three different types of mills was
examined based on the measurements of DC. The results showed that the ball mill
induced significantly the least destruction to the collagen structure in comparison to the
micro-dismembrator and milling machine. Interestingly, the damage caused by micro-
dismembrator and milling machine is reversible after 1-week storage at 8 °C. It is
expected that the triple helix is unfolded but the polypeptide chains are still fixed in their
positions, which enable the recovery of the native triple helix by building hydrogen
bonds.
Summary
III
The thermal stability of the collagenous tissues was considered as a crucial assessment
parameter because it is sensitive to any structural destruction or modification. The
thermal denaturation of collagen induces unfolding of triple helix into random coils by
breaking the hydrogen bonds; the ability of collagen to resist this unfolding is an
indication of its “healthiness”. The tissues were treated thermally in the range of (55-200
°C) for 1 h in furnace and then incubated with α-chymotrypsin to determine the fraction
of denatured collagen (DC).
The DC for Tutoplast-processed bovine cancellous bone remained unchanged till the
temperature 90 °C, and then it started to increase linearly with increasing temperature.
Regarding the thermal stability of bovine pericardium, the measurements of DC showed
higher thermal stability of the native lyophilized (initial water content 7%) and Tutoplast-
processed pericardium (initial water content 1.7%) in comparison with the native
pericardium (initial water content 85%). The DC for native lyophilized and Tutoplast-
processed pericardium remained unchanged until 135 and 150 °C respectively, whereas
for the native pericardium, it started to increase from 55 °C. This could be attributed to
the water content, according to the polymer in a box mechanism; dehydration reduces the
lateral dimensions of the lattice, constrains the number of possible configurations,
reduces the free-volume available for denaturating α-chains, reduces the configuration
entropy and thereby increases the thermal stability of collagen.
A reduction of DC at high temperatures (185-200 °C) has been observed with the
Tutoplast-processed and the lyophilized pericardium but not with the native pericardium.
This could be attributed to the formation of heat generated advanced glycation end
products (AGEs), which hinders the enzymatic digestion of collagen. The
spectrophotometric measurements of the extent of browning proved the formation of
AGEs. The absence of AGEs with the native pericardium could be ascribed to the higher
moisture content that delays the formation of AGEs.
The dominance of water content during the measurements of DC made it necessary to
exclude the effect of the water content by performing measurements of isotonic shrinkage
temperature at fully hydrated conditions in water bath, in which only the structure
integrity and healthiness plays a role in shaping the thermal stability.
Summary
IV
It was seen obviously the Tutoplast-processed pericardium has lower thermal stability or
thermoelasticity to resist the thermal shrinkage as indication of structural modification or
destruction caused by the processing. The Tutoplast-processed started to shrink from 42
°C, whereas the native lyophilized and the native pericardium from 64 and 65 °C
respectively. Furthermore, the shrinkage process of Tutoplast-processed strips was slow
and took place over relatively wide temperature range (42-70 °C), in comparison to that
of the lyophilized and the native pericardium that took place with 5 degrees. The
presence of residual ions in the Tutoplast-processed pericardium resulted from the
processing, which was confirmed by the measurements of the conductivity, resulted in
swelling of the tissues during the shrinkage process and consequently to thick samples,
which shrunk too slowly.
The analysis of the DC and shrinkage temperature measurements draws the conclusion
that the Tutoplast-process induces two contradictory factors, stabilizing factor,
represented by the dehydration that dominant under dry conditions, and destabilizing
factor, which may represented by structural modification that becomes visible under fully
hydrated conditions. The next challenge was to investigate the role or the effect of each
step of Tutoplat process and its contribution in the structural modification caused by the
process.
The sodium hydroxide treatment in the process is used as a protection against creutzfeldt-
Jakob disease and is scientifically recognized as an acceptable and effective methodology
for reducing prion infectivity by six log. The effect of the sodium hydroxide treatment on
the stability of bovine pericardium process was examined. It was proved the amount of
dissolved or hydrolyzed collagen after 150 min 1 N NaOH treatment at room temperature
was not significant and lower than 1 % of the original dry weight this could be attributed
to the fact that NaOH treatment doesn’t destroy the helical structure of collagen. It was
shown that 1 N NaOH treatment induces swelling of the pericardium tissues,
modification of some amino acids and destruction of intra-and intermolecular collagen
cross-links leading to significantly lower shrinkage temperature, approximately 25
degrees lower than the native untreated samples. Furthermore the shrinkage of the
NaOH-treated strips was too slow and occurred over wide temperature range,
Summary
V
approximately 30 degrees, because the samples are thick and swelled from the action of
the alkali, which prevented smooth shrinkage of the samples.
In order to treat or to remove the effect caused by the NaOH treatment, several treatment
possibilities have been tested. The aim of this treatment was to restore the pericardium
strips to their physiological state and pH. The volume of NaOH as well as of CH3COOH
was chosen to have dully submerged strips, for example, for the treatment of 10 and 5
strips 100 and 50 ml were used respectively. It is seen that the 1 N CH3COOH treatment
shifts the pH value of the strips too rapidly from the basic to the acidic region causing
damage also. 5 and 15 min treatment was enough to shift the pH to 5 and 3 respectively
yielding pericardium strips with thermal shrinkage starting at 50 and 45 °C respectively.
Therefore it was reasonable to test the effect of lower concentrations of CH3COOH. The
15-min treatment with 0.1 N CH3COOH shifted the pH value of the pericardium strips to
6, resulting in thermal shrinkage starts at 62 °C, which was close to that of the native
untreated pericardium.
Also as alternative to the acid treatment, the efficiency of different washing or rinsing
fluids, such as distilled water and phosphate buffer (pH 7.4) has been tested. It is
observed that submerging or washing the NaOH-treated samples once with water or
phosphate buffer is not sufficient to reduce the pH value of the samples due to the limited
capacity of the water or buffer to wash the ions. A complete neutralization of the samples
could be achieved by intensive washing with distilled water baths for 90 min, in which
the water bath has to be changed every 10 or 15 min.
The best variant has been achieved by treating the NaOH treated strips with 1 N
CH3COOH for 15 min followed by one or two 10-min distilled water washing bath. With
this variant, the pericardium strips has a final pH value 8 and a thermal shrinkage starts at
64 °C.
The measurements of DC show no significant influence of the NaOH treatment. This
could be attributed to the fact that α-chymotrypsin is not active to digest collagen at
highly alkaline conditions and only active at neutral to slightly alkaline ranges
The hydrogen peroxide step in the process has been found to be effective against the
human immunodeficiency virus (HIV). Through this treatment, soluble proteins are
Summary
VI
eliminated, remaining viruses are inactivated and the potential for graft rejection is
minimized. The effect of H2O2 on the stability of the pericardium was studied.
It was seen that the treatment with 3% H2O2 (pH = 5.52) is almost not destructive to the
collagen structure. This was confirmed by the high thermal stability assessed by the
measurements of isotonic shrinkage temperature. The samples treated with 10% H2O2
(pH = 4.13) had almost similar thermal stability to those treated with 3% H2O2. In
contrast to the 3 and 10% treatment, the treatment with 30% (pH = 2.40) was extreme
destructive and resulted in completely destructed pericardium strips, which can’t be
further tested with the isotonic shrinkage technique. Hydrogen peroxide is a relatively
nonspecific oxidizing agent, under acidic conditions the primary reaction is the
conversion of methionine residues to sulfoxide. Oxidation of Met residues is associated
with the loss of the biological activity for many proteins. It is expected the treatment with
H2O2 under mild concentrations and pH doesn’t lead to complete oxidation of the Met
residues and consequently to the complete destruction of the collagen, which was
observed at extreme concentration and pH.
Regarding the DC measurements, as discussed with the NaOH treatment, α-chymotrypsin
couldn’t attack the tissues under acidic conditions because it is inactive in this pH range.
Acetone treatment is used in Tutoplast process to inactive the remaining prion and
viruses and to dehydrate the tissues. The effect of acetone treatment on the stability of
pericardium was examined. In the current study the pure acetone was compared with the
graded acetone. After 18 days, both of them have almost the same weight loss and
shrinkage, however with different curve course. The pure acetone is much more effective
to dehydrate the tissues; the maximum weight loss has almost been achieved within the
first two days, whereas the maximum weight loss has been reached at the tenth day
during the graded acetone treatment.
Regarding the shrinkage, during the first 2 days of the pure acetone treatment, the tissue
shrunk 22.68% of their volume. Further shrinkage was also observed until the maximum
shrinkage reached at the sixteenth day, 52.91%.
The functions of the acetone treatment in the Tutoplast-process are tissue dehydration and
inactivation of any prions and viruses. Therefore it is recommended to check if the 2-day
pure acetone treatment is sufficient to inactivate prions and viruses.
Summary
VII
The acetone treatment has no significant influence on the collagen denaturation. The DC
values of pure acetone as well as graded acetone treated samples are almost similar to
those of the native untreated samples.
The ideal grafting material should not only be adequately osteogenic, -conductive, and -
inductive but also mechanically stable and disease free. Therefore the effect of the
processing on the mechanical properties of the tissues was examined.
The analysis of ultimate strength and elastic modulus values after thermal treatments of
bone cubes gives no clear relationship or statement about the temperature-dependency of
the mechanical properties of the bone samples. The influence of the structure
heterogeneity (non-uniform mineral distribution) and the fiber orientation could be
behind the scattering of the results and the absence of convenient statement about the
mechanical properties of bones.
The analysis of the mechanical properties of pericardium did not lead to any conclusion
about the influence of the processing of the mechanical stability. It was expected, despite
the separation between the left and the right side of the sac, that the effect of anisotropy
was dominant over the influence of the processing. It can be concluded that only SALS-
selected samples can be used to assess the effect of the processing on the mechanical
properties of the pericardium.
Nomenclature
VIII
Nomenclature Symbol Description Units
G∆ Gibbs free energy kJ/mol
H∆ van’t Hoff enthalpy kJ/mol
S∆ Entropy of transition kJ/mol.K
A The frequency factor sec-1
B Heating rate parameter -
DC Fraction of denatured collagen %
E The activation energy kJ/mol
k The kinetic first order constant sec-1
K The equilibrium constant -
/K The apparent first order constant sec-1
L The length of the fiber or tissue mm
0L The initial length of the fiber mm
∞L The length of the completely shrunken fiber mm
l The length of the collagen molecule mm
Dl The length of the denatured collagen molecule mm
Nl The length of the native collagen molecule mm
AAm Mass of aromatic amino acids µg
Cm Collagen mass µg
n The number of collagen molecules -
Nn The number of native collagen molecules -
Dn The number of denatured collagen molecules -
q The heating rate °C/min
R The ideal gas constant J/K. mol
2/1t The time of half shrinkage sec
∞t The time of maximum shrinkage sec
x Temperature parameter -
UX The fraction of unfolded collagen %
Nomenclature
IX
Symbol Description Units
FX The fraction of final denatured collagen %
NX The fraction of native collagen %
SX The fraction of collagen in the supernatant %
z Adaptation parameter -
Abbreviations Symbol Description
AGEs Advanced Glycation End Products
BSE Bovine Spongiform Encephalopathy
CEL Carboxyethyl)lysine
CJD Creutzfeldt - Jakob disease
CML Carboxymethyllysine
DBM Demineralized Bone Matrix
DHLN Dihydroxylysinonorleucine
DSC Differential Scanning Calorimetry
Gly Glycine
HA Hydroxyapatite
HIV Human Immunodeficiency Virus
HLKNL Hydroxylysino-5-ketonorleucine
HLN Hydroxylysinonorleucine
HP Hydroxylysyl Pyridinoline
Hyp Hydroxyproline
LKNL Lysino-5-ketonorleucine
LP Lysyl Pyridinoline
MOLD Methylglyoxal-Lysine Dimer
PE Polyethylene
PTFE Polytetrafluoroethylene
rER Rough Endoplasmic Reticulum
Table of contents
X
1 Introduction................................................................................................................. 1
2 State of the art ............................................................................................................. 2
2.1 Bone grafting ...................................................................................................... 2
2.1.1 Graft types................................................................................................... 2
2.1.1.1 Autograft ................................................................................................. 2
2.1.1.2 Allograft.................................................................................................. 2
2.1.1.3 Xenograft ................................................................................................ 3
2.1.1.4 Alloplastic ............................................................................................... 3
2.1.2 Bone healing ............................................................................................... 4
2.1.3 Graft processing .......................................................................................... 6
2.1.3.1 Graft Processing Techniques used in the Medical Field......................... 6
2.1.3.2 Tutoplast® process ................................................................................. 7
2.2 Collagen ............................................................................................................ 10
2.2.1 Collagen types........................................................................................... 10
2.2.2 Collagen Synthesis.................................................................................... 12
2.2.3 Collagen Cross-links................................................................................. 14
2.2.4 The role of hydroxyproline in collagen stabilization................................ 16
2.2.5 The Thermal stability of collagen ............................................................. 18
2.2.6 Advanced Glycation End Products (AGEs).............................................. 19
2.3 Bone .................................................................................................................. 20
2.3.1 Bone Composition .................................................................................... 20
2.3.2 Bone Hierarchy ......................................................................................... 21
2.4 Pericardium....................................................................................................... 23
3 The Objectives .......................................................................................................... 26
4 Materials and Methods.............................................................................................. 27
4.1 Materials ........................................................................................................... 27
4.1.1 Bovine Bones ............................................................................................ 27
4.1.2 Bovine Pericardium .................................................................................. 28
4.2 Methods............................................................................................................. 29
4.2.1 Preparation Steps ...................................................................................... 29
Table of contents
XI
4.2.1.1 The pulverization of the bones.............................................................. 29
4.2.1.2 The Demineralization of Bones ............................................................ 30
4.2.2 The Determination of Denatured Collagen (DC)...................................... 30
4.2.2.1 A Selective Digestion Method .............................................................. 31
4.2.2.2 Spectrophotometeric Determination of the DC .................................... 32
4.2.3 The Measurements of the Extent of Browning ......................................... 34
4.2.4 The Measurements of the Isotonic Shrinkage temperature....................... 34
4.2.5 SDS-PAGE ............................................................................................... 35
4.2.6 Characterization of the Mechanical Properties ......................................... 39
4.2.7 The measurements of the Thermal Conductivity...................................... 39
4.3 Different physical and chemical treatment ....................................................... 39
4.3.1 The thermal treatment of collagenous tissues........................................... 40
4.3.1.1 The thermal treatment of bovine bone .................................................. 40
4.3.1.2 The thermal stability of bovine pericardium......................................... 40
4.3.2 The Sodium hydroxide treatment and the corresponding neutralization .. 41
4.3.2.1 Sodium hydroxide treatment................................................................. 41
4.3.2.2 The neutralization of the tissues after the NaOH treatment.................. 42
4.3.3 The hydrogen peroxide treatment ............................................................. 43
4.3.4 The acetone treatment ............................................................................... 43
4.3.5 The determination of water content .......................................................... 44
5 Results Interpretations .............................................................................................. 45
5.1 The Effect of Bone Pulverization on the Collagen ........................................... 45
5.1.1 The Measurements of DC ......................................................................... 45
5.1.2 Discussion of the Results .......................................................................... 46
5.2 The Thermal stability of Collagenous Tissues.................................................. 47
5.2.1 Analysis of the Thermal Stability with the Measurements of DC............ 47
5.2.1.1 The Thermal Stability of Tutoplast-Processed Bovine Bone ............... 47
5.2.1.2 The Thermal Stability of Native Bovine Pericardium .......................... 48
5.2.1.3 The Thermal Stability of Tutoplast-Processed Bovine Pericardium .... 49
5.2.1.4 The Thermal Stability of the Lyophilized Bovine Pericardium............ 50
5.2.1.5 The Measurements of the Extent of Browning ..................................... 51
Table of contents
XII
5.2.1.6 Discussion of the Results ...................................................................... 52
5.2.1.7 Modeling of the Thermal Denaturation of Pericardium ....................... 59
5.2.2 Measurements of Isotonic Shrinkage Temperature .................................. 65
5.2.2.1 The Results............................................................................................ 66
5.2.2.2 Discussion of the Results ...................................................................... 66
5.2.2.3 Modelling of the Thermal Shrinkage of Pericardium........................... 68
5.2.3 SDS-PAGE investigations ........................................................................ 78
5.2.3.1 Results................................................................................................... 78
5.2.3.2 Discussion ............................................................................................. 81
5.3 The Effect of Different Steps in the Tutoplast® Process.................................. 83
5.3.1 The Effect of Sodium Hydroxide Treatment ............................................ 83
5.3.1.1 The Hydrolysis of Collagen Amino Acids............................................ 84
5.3.1.2 The Measurements of the Shrinkage Temperature ............................... 86
5.3.1.2.1 The effect of NaOH Solution.......................................................... 86
5.3.1.2.2 The Effect of the Neutralization Step ............................................. 87
5.3.1.3 The Measurements of DC ..................................................................... 91
5.3.1.4 SDS-PAGE Investigations .................................................................... 92
5.3.1.5 Discussions ........................................................................................... 94
5.3.2 The Effect of Hydrogen Peroxide Treatment............................................ 98
5.3.2.1 The Shrinkage Temperature Measurements ......................................... 98
5.3.2.2 Measurements of DC ............................................................................ 98
5.3.2.3 SDS-PAGE Investigations .................................................................... 99
5.3.2.4 Discussion of the Results .................................................................... 100
5.3.3 The Influence of Acetone Treatment ...................................................... 101
5.3.3.1 The Extent of Drying and Shrinkage .................................................. 102
5.3.3.2 The Measurements of DC ................................................................... 103
5.3.3.3 Discussion of the Results .................................................................... 104
5.4 The Mechanical Properties of the Collagenous Tissues ................................. 105
5.4.1 The Mechanical Properties of Bovine Bones.......................................... 105
5.4.2 The Mechanical Properties of Bovine Pericardium................................ 106
5.4.3 Discussion of the Results ........................................................................ 107
Table of contents
XIII
6 Optimization of Tutoplast Process.......................................................................... 110
6.1 The Sodium Hydroxide treatment................................................................... 110
6.2 The Hydrogen Peroxide treatment .................................................................. 111
6.3 The Acetone treatment.................................................................................... 111
7 Literature................................................................................................................. 112
1. Introduction
1
1 Introduction Transplantation has become world wide one of the most important innovations in the
medical field. Organs save lives and tissues improve the quality of the life for millions of
people. A single donor can help more than 50 persons awaiting an organ or transplant.
Bone and tissue donations give a new meaning to life for many patients each year. While
most people are familiar with organ donor programs, tissue donation is a relatively new
concept.
Various grafting material including autografts, allografts, xenografts and synthetic
materials have been used clinically. The autograft is considered as the gold-standard graft
because it contains viable cells and growth factors, which stimulate the healing of the
graft. However, the limited availability and the additional morbidity are the main
disadvantages associated with the transplantation of autografts. Therefore, alternative
grafting materials have been always used to fulfill the increasing demand for grafts in the
medical field.
Allografts are the surgeon’s second choice, however the concern about the
immunological reaction and the transmission of host diseases are the main limitation
coupled with the use of Allografts.
Several preprocessing treatments have been applied to deal with allografts limitations.
However, achieving the two goals, suppressing the immunological reactions and avoiding
the transmission of host diseases, requires a combination of different preprocessing
treatments. It was proved that the preprocessing treatments could destruct the biological
and structural integrity of the tissues.
Tutoplast® process is a comprehensive and validated conservation and sterilization
process, been used for 30 years, aims to eliminate the antigenicity of allografts as well as
the possibility of disease transmission without affecting the mechanical and biological
properties of the tissues. The process deactivates, destroys and removes all unwanted
materials, such as fats, cells, viruses and microbes.
In this dissertation, the influence of the Tutoplast® process on the thermal and
mechanical properties of the tissues have been assessed in order to establish a
background profile for the possible future plans of the process optimization.
2. State of the Art
2
2 State of the art 2.1 Bone grafting Bone grafting is a surgical procedure where the bone is taken from donor site and
implanted into the patient to improve the function or strengthen a damaged part. Bone
graft is the second most common transplantation tissue after blood (Boyce T 1999). More
than 500,000 bone grafting procedures are happening annually in the United States and
2.2 million worldwide in order to repair bone defects in orthopaedics, neurosurgery and
dentistry (Lewandrowski, D. Gresser et al. 2000). Bone-grafting is usually required to
stimulate bone-healing. In addition, spinal fusions, filling defects following removal of
bone tumors and several congenital diseases may require bone grafting (Giannoudis,
Dinopoulos et al. 2005).
2.1.1 Graft types
There are four classes of bone grafts (Hoexter 2002): autograft, allograft, xenograft and
alloplastic.
2.1.1.1 Autograft
The autograft is defined as the tissue transplanted from one site to another within the
same individual. The gold standard of bone-grafting is harvesting autologous cortical and
cancellous bone from the iliac crest (Giannoudis, Dinopoulos et al. 2005). Although
autologous bone can be harvested from the tibia, fibula, olecranon, distal radius, and ribs,
the iliac crest remains the most common donor site (Laurie, Kaban et al. 1984). Despite
the best success rates in bone grafting achieved with autografts, harvesting autologous
bone from the iliac crest has, however, several downsides as it lengthens the overall
surgical procedure and is usually complicated by residual pain and cosmetic
disadvantages (Summers and Eisenstein 1989; Giannoudis, Dinopoulos et al. 2005).
Furthermore, it may fail in clinical practice as most of the cellular (osteogenic) elements
do not survive transplantation (Sandhu, Grewal et al. 1999).
2.1.1.2 Allograft
Allografts are tissues taken from individuals of the same species as the hosts. Allograft is
the most frequently chosen bone substitute and is regarded as the surgeon's second option
(Carter 1999). Its use has increased 15-fold during the past decade and accounts for about
one-third of bone grafts performed in the United States (Boyce T 1999). The concern
2. State of the Art
3
about the immunological response and the transmission of host diseases is the main
obstruction to use allografts. The processing of allograft tissue lowers the risk of
transferring viral diseases but, that can significantly deteriorate the biological and
mechanical properties of the graft (Bos, Goldberg et al. 1983; Giannoudis, Dinopoulos et
al. 2005).
2.1.1.3 Xenograft
The xenograft is defined as a tissue taken from other species (i.e. bone of bovine origin).
Like allografts, xenografts are available in large amounts but have to be preprocessed to
avoid the immunological reactions.
2.1.1.4 Alloplastic
Alloplastic materials, synthetic materials, offer the potential for "off-the-shelf" solutions
to reconstructive tissue needs (replacement and/or augmentation), which avoid donor
scars and morbidity and typically simplify the operative procedure in terms of time and
complexity of technique (Eppley 1999). The wide range of implantable synthetic
materials can be divided into a few categories: carbon-based polymers, non–carbon-based
polymers, metals, and ceramics (MacRae 2005). The chemical composition, physical
form, and differences in surface configuration result in varying levels of bioresorbability
(Hoexter 2002). MacRae (MacRae 2005) has evaluated the application of alloplastic
grafts:
Non-carbon-based polymers
The silicone, as a non–carbon-based polymer, is highly resistant to degradation and has a
high degree of chemical inertness due to its silicon-oxygen bonds. Silicone implants
retain their strength and flexibility though a wide range of temperatures and easily can be
sterilized. However, silicone has a tendency to fragment and deteriorate, when subjected
to repeated movement with mechanical loading. The solid silicone is considered as inert,
however, in the liquid or gel form, the silicone is not as inert and can incite a chronic
inflammatory reaction. As with other alloplastic implants, one complication of silicone
implants is extrusion.
Carbon-based polymers
Carbon-based polymers have been used in various forms and include
polytetrafluoroethylene (PTFE), polyethylene (PE), aliphatic polyesters, and
2. State of the Art
4
methylmethacrylate. PTFE is nonresorbable and highly biocompatible with no tendency
for chronic inflammatory reaction, whereas the aliphatic polyesters are used extensively
for the advantage of being resorbable. In general, the surgical requirement or need
determine if the material should be resorbable or not.
Metals
Metals have been used in maxillofacial plating for their strength and durability. Stainless
steel no longer is used due to its tendency to corrode. Titanium, an elemental metal, has
had no reports of allergy, toxicity, or tumorigenesis.
Ceramics
Hydroxyapatite (HA) is calcium phosphate salt, the principal inorganic compound in
bone matrix. It can be produced synthetically in a dense form, but this dense form of HA
was found to be difficult to shape and prone to migration and extrusion. Porous forms of
HA have been much more successful and are based on the calcium carbonate structure of
marine corals. Their porosity permits fibrovascular and osseous ingrowth. However, HA
implants cannot tolerate significant load bearing and tend to crack and fracture.
One major limitation of alloplastic implants is their susceptibility to infection. A variety
of factors contribute to this risk, including the location of the implant, vascularity of the
pocket, operative technique in handling and placing the implant, and the ability of
bacteria to adhere to and penetrate the material.
In general, it is not easy to decide which graft type is better; the decision must be taken
for each case separately depending on the surgical requirement and the patient medical
history.
2.1.2 Bone healing
Bone is unique tissue because it is continuously metabolically active and thus subject to a
variety of systemic and local factors throughout life (Pilitsis, Lucas et al. 2002). Bone
consists of the following bone cells (Felsenberg 2001; Pilitsis, Lucas et al. 2002):
osteoblasts, osteoclast and osteocytes, which are essential for the bone formation process
Osteoblasts
Osteoblasts or the bone-forming cells synthesize and secrete unmineralized ground
substance, the osteoid, and found in areas of high metabolism within the bone (Van De
Graaff 1998). Osteoblasts are involved in matrix development as well as calcification [3],
2. State of the Art
5
As the process of bone deposition ends, some osteoblasts remain on the periosteum and
endosteum, whereas others become osteocytes (Kalfas 2001).
Osteoclasts
Osteoclasts or the bone resorbing cells are very important for bone growth, healing, and
remodeling. Osteoclasts secrete proteolytic enzymes that able to dissolve both the
inorganic and organic osseous matrices, resulting in the formation of erosive pits called
Howship lacunae and the release of calcium and phosphate (Kalfas 2001).
Osteocytes
Mature bone cells, made from osteoblasts, maintain healthy bone tissue by secreting
enzymes and controlling the bone mineral content. They also control the calcium release
from the bone tissue to the blood.
The ability of bone healing and fusions formation by transplantation of bone grafts is
based on three key concepts (Pilitsis, Lucas et al. 2002): osteogenesis, osteoconduction
and osteoconduction.
Osteogenesis
Osteogenesis, defined as the ability to produce new bone, is determined by the presence
of osteoprogenitor cells and osteogenic precursor cells in the area. Both fresh autografts
and bone marrow cells contain osteogenic cells.
Osteoconduction
Osteoconductive properties are determined by the presence of a scaffold that allows for
vascular and cellular migration, attachment, and distribution (Helm, Dayoub et al. 2001).
Osteoconduction may be achieved through the use of autografts, allografts, demineralized
bone matrix (DBM), hydroxyapatite, and collagen.
Osteoinduction
Osteoinduction is defined as the ability to stimulate stem cells to differentiate into mature
cells through stimulation by local growth factors (Subach, Haid et al. 2001). Bone
morphogenetic proteins and DBM are the most potent osteoinductive materials, although
allo- and autografts have some osteoinductive properties (Kalfas 2001).
2. State of the Art
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The terms osteogenic, osteoconductive, and osteoinductive are not absolute, and are best
understood when used in the context of a comparative study (Bauer and Muschler 2000).
The ideal grafting material should be not only osteogenic, -conductive, and -inductive but
also mechanically stable and disease free (Kalfas 2001; Pilitsis, Lucas et al. 2002)
Autografts possess these properties. However, their use is limited by the morbidity
associated with the process of obtaining them and by the often-insufficient amount of
graft (Helm, Dayoub et al. 2001). Allografts are osteoconductive, weakly osteoinductive,
not osteogenic (Pilitsis, Lucas et al. 2002).
2.1.3 Graft processing
As mentioned in the previous section, the use of allograft and xenograft is restricted by
the concern about the immunological reactions and the transmission of host diseases.
Bone allografts are different from most solid organ transplants in that cells are removed
intentionally as thoroughly as possible to minimize immunologic rejection (Bauer and
Muschler 2000). Many experimental studies have shown that in general, bone graft
materials show optimum incorporation with the host when histocompatibility differences
are minimized by either matching tissue types, or processing the allografts with
techniques that reduce immunogenicity (Bonfiglio and Jeter 1972; Blives 1975;
Goldberg, Powell et al. 1985). The osteoinductive, osteoconductive, and biomechanical
properties of different allografts vary depending on the methods of graft processing
(Bauer and Muschler 2000; Hofmann, Konrad et al. 2005).
2.1.3.1 Graft processing techniques used in the medical field
Several studies showed that deep-frozen or freeze-dried allogeneic tendons could be used
without invoking an immunological reaction (Shino, Inoue et al. 1988; Horibe, Shino et
al. 1991; Toritsuka, Shino et al. 1997). However, the transmission of viral diseases with
allografts processed by these methods is not excluded (Fideler, Vangsness et al. 1994).
Regarding the risk of disease transmission, such as hepatitis and human
immunodeficiency virus (HIV), a careful donor-screening has to be performed but some
diseases can be undetectable during the incubation period (Steelman 1994). Therefore
secondary sterilization, such as γ-irradiation is desirable for safer clinical use of allografts
(Toritsuka, Shino et al. 1997). Unfortunately high levels of gamma irradiation are
required to inactivate viral contamination. Fideler et al. (Fideler, Vangsness et al. 1994)
2. State of the Art
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found that some HIV-infected human patellar tendon-bone allografts remained infected
when irradiated to levels less than 3 Mrad (30,000 Gy). Several studies showed that deep
freezing as well as freeze drying cause no significant change in the mechanical integrity
of the graft (Pelker, Friedlaender et al. 1984; Woo, Orlando et al. 1986; Paulos, France et
al. 1987), whereas γ-irradiation generates free radicals creating deterioration in the
mechanical properties, especially in a dried condition (Cheung, Perelman et al. 1990;
Maeda, Inoue et al. 1993; Salehpour, Butler et al. 1995). Dose-dependent destruction
caused by gamma sterilization was illustrated (Gibbons, Butler et al. 1991). The
allografts were not significantly affected by 2 Mrad of gamma irradiation but weakened
significantly after 3 Mrad of irradiation. It was also confirmed that freeze dying followed
by gamma irradiation reduces the tensile strength of patellar tendons significantly,
whereas reversing the order, gamma irradiation followed by freeze drying, changes the
tensile strength minimally (Haut and Powlison 1989). This suggests that irradiation of dry
tissue causes severe changes in the mechanical properties due to reduced protection
against the action of oxygen and the lack of water which absorbs energy (Maeda, Inoue et
al. 1993).
2.1.3.2 Tutoplast® process
The Tutoplast® process has been available for over 30 years to sterilize and preserve
tissues utilized in all surgical disciplines, including dentistry, neurosurgery, orthopedics,
ophthalmology, otolaryngology, gynecology, urology and pediatric surgery (Schoepf
2006). In contrast to the deep freezing and freeze drying, which decrease the viral titers
but do not guarantee complete eradication (Jackson, Grood et al. 1988), Tutoplast®
process is a comprehensive sterilization and preservation method that aims to eliminate
the antigenicity of allograft as well as the possibility of disease transmission without
affecting the mechanical and biological properties of the tissues. It was reported that the
human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other viruses are
inactivated during Tutoplast® process (Diringer and Braig 1989; Deinhardt 1991;
Koschatzky and Wolfel 1992). Furthermore, neither viral transmission during clinical use
nor immune response during experimental study has occurred using Tutoplast®
processed dura matter (Stoess and Pesch 1977). Regarding the biomechanical analysis, it
was reported that Tutoplast® processed fascia lata has significantly higher stiffness than
2. State of the Art
8
freeze dried one (Hinton, Jinnah et al. 1992). Maeda et al (Maeda, Inoue et al. 1993) have
examined the effect of gamma irradiation on canine tendons before Tutoplast® process
and after the process. They showed that irradiation before Tutoplast® process affected
the mechanical properties minimally, whereas reversing the order had deep effects on the
mechanical properties.
The process consists of different main steps; the whole process is shown in fig. 2.1:
Figure 2.1: Tutoplast® Process for soft and hard tissues
Processing steps
Donor screening
Delipidization
Osmotic treatment
NaOH treatment
H2O2 oxidation
Acetone dehydration
Primary Packaging
γ sterilization
Hard tissues Soft tissues
Final packaging
Tutoplast® Process
2. State of the Art
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Delipidization
Lipids are removed in an ultrasonic acetone bath. Lipids removal is important because
they may interfere with healing process and form cytotoxic product when irradiated
(Moreau, Gallois et al. 2000). This step also prepares the tissue so that the subsequent
steps could penetrate the graft more effectively.
Osmotic treatment
In this step a series of alternating hypertonic saline and distilled water baths are used.
This step ruptures the cell membrane, kills bacteria, washes out cellular debris and
removes antigens.
Hydrogen peroxide treatment
This step has been found to be effective against the human immunodeficiency virus
(Hinton, Jinnah et al. 1992). Hydrogen peroxide is a relatively nonspecific oxidizing
agent, which reacts with a wide variety of organic compounds. It can modify thioether,
indole, sulfhydryl, disulfide, imidazole and phenolic at the neutral or slightly alkaline
conditions (Manning, Patel et al. 1989). Under acidic conditions the primary reaction is
the conversion of methionine residues to sulfoxide (Neumann and Timasheff 1972).
Oxidation of Met residues is associated with the loss of the biological activity for many
proteins (Manning, Patel et al. 1989). Through this treatment, soluble proteins are
eliminated, remaining viruses are inactivated and the potential for graft rejection is
minimized
Acetone dehydration
The acetone wash, followed by vacuum extraction, dehydrates the tissues allowing them
to be room temperature storable. During this step any residual prions are removed and
enveloped viruses are inactivated.
Sodium hydroxide treatment
The soft tissues, not bones, are received 1 N NaOH for 1 h at room temperature to reduce
the prion infectivity as protection step against Bovine Spongiform Encephalopathy (BSE)
and Creutzfeldt - Jakob disease (CJD). It was confirmed that this step reduces prion
infectivity by six log (Brown, Rohwer et al. 1986).
2. State of the Art
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After Tutoplast® process, tissues are cut, packed and sterilized using limited-dose
gamma irradiation (17.8-25 kGy) to eliminate any microbial contaminations that may
result from handling and packaging.
2.2 Collagen Collagen is derived from the Greek words kolla and gennan, meaning to produce glue.
The collagens constitute a family of related proteins that are assembled in a variety of
supramolecular structures in extracellular matrices. These structures illustrate how the
basic motif of collagens was utilized to generate a diversity of supramolecular matrix
network to accomplish an equally diverse number of functions in the tissues of
multicellular organisms (Vuorio and De Crombrugghe 1990). Collagen, a fibrous protein,
is the most abundant protein in animals, accounting for 30 % of all proteins in mammals
(Patino, Neiders et al. 2002). Collagen is the major protein of the bone, teeth, tendon,
ligaments, cornea and cartilage. It is composed of three chains and has a unique Gly-X-Y
repeating sequence, where X and Y are frequently proline and hydroxyproline
respectively. The chemical characterization of collagen was an impasse for many years
because of the insolubility of collagen fibers. The breakthrough came when it was found
that collagen from the tissues of young animals can be extracted in soluble form because
it is not yet cross-linked (Stryer 1988).
2.2.1 Collagen types
There are 20 collagen types, type I is the most abundant structural protein found in
vertebrate. Based on their supramolecular structures, the collagens are divided into two
main classes (Vuorio and De Crombrugghe 1990; Patino, Neiders et al. 2002): fibril-
forming (or fibrillar) collagens and non-fibril-forming collagens. The fibrillar collagens,
contain type I, II, III, V and XI collagens, form highly organized fibers and fibrils
providing the structural support for the body in skeleton, skin, blood vessels, nerves and
the fibrous capsules of organs (Vuorio and De Crombrugghe 1990). The non-fibril
forming collagens, all the collagen falling outside the fibril-forming collagens, are very
heterogeneous structurally and functionally and have been further classified according to
their molecular characteristics, supramolecular structures, and the types of extracellular
networks that they form into: basement membrane collagens, short chain collagens and
fibril-associated collagens (Hulmes 1992; Patino, Neiders et al. 2002). A list of the
2. State of the Art
11
collagen types, their constituent α-chains, and the tissue distribution is presented in Table
2-1.
Table 2-1: Collagen types (modified from (Patino, Neiders et al. 2002))
Type α chains Tissue Distribution
I α1(I), α2(I) Connective tissues (bone,
tendon, skin, etc)
II α1(II) Cartilage
III α1(III) Extensible connective
tissues (skin, lung)
IV α1(IV), α2(IV), α3(IV),
α4(IV), α5(IV)
Basement membranes
V α1(V), α2(V), α3(V) Tissue containing collagen
I, minor component
VI α1(VI), α2(VI), α3(VI) Most connective tissues,
including cartilage
VII α1(VII) Basement membrane
associated anchoring fibrils
VIII α1(VIII), α2(VIII) Product of endothelial and
various tumor cell lines
IX α1(IX), α2(IX), α3(IX) Tissue containing collagen
II, minor component
X α1(X) Hypertrophic zone of
cartilage
XI α1(XI), α2(XI), α3(XI) Tissue containing collagen
II, minor component
XII α1(XII) Tissue containing collagen
I, minor component
XIII α1(XIII)
XIV α1(XIV) Tissue containing collagen
I, minor component
2. State of the Art
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Type I collagen is the most abundant animal protein, covers 80% to 99% of total collagen
(Burgeson and Nimni 1992), and forms the matrix of bone, skin, pericardium and other
collagenous tissues. Type I collagen molecules are composed of three polypeptide chains,
two identical α I chains and one α II, each consists of 1300-1700 amino acid residues, the
majority (~ 1000) are organized into a central triple helix configuration (Shirley and
Boot-Handford 1998; Wright and Humphrey 2002). Mutations in type I collagen,
deletions, insertions, and single amino acid substitution, result in many diseases, such as
osteogenesis imperfecta, Ehlers Danlos syndromes and many degenerative diseases
(Patino, Neiders et al. 2002)
2.2.2 Collagen synthesis
Intracellular events
Procollagen molecules have been identified as the precursors of collagen molecules
(Burgeson and Nimni 1992). The synthesis of procollagen molecules takes place within
the fibroblast. The first step in the process of collagen synthesis is the formation of
collagen specific messenger-RNA (Vuorio and De Crombrugghe 1990). After the gene
transcription, functional mRNA are formed and transported to the cytoplasm and
translated on membrane-bound polysomes to the rough endoplasmic reticulum (rER)
(Burgeson and Nimni 1992). As the collagen polypeptides are synthesized in the rER,
important post-translational events accompany this process. Prolyl and lysyl hydroxylases
mediate the hydroxylation of proline and lysine. Thereafter glycosylation takes place, the
hydroxylysine residues are covalently bound to carbohydrate units (Stryer 1988).
Glycosylations are catalyzed by two specific enzymes, a galactosyl transferase and
glucosyl transferase. The importance of post-translational modification of the collagen is
illustrated in heritable disorders of collagen. In Ehlers-Danlos syndrome type IV, the
hydroxylation is reduced causing dysfunctions of the connective tissue, conversely,
overhydroxylation results in osteogenesis imperfecta. Once the hydroxylation and
glycosylation are completed, the individual α-chains align to form the triple helix. After
the formation of triple helix, the procollagen molecules move from rER towards Golgi
apparatus. In the Golgi, procollagen molecules are packed in Golgi-derived vesicles and
carried toward the cellular membrane by cytoskeletal movements.
2. State of the Art
13
Extracellualr events
Once the newly synthesized procollagen molecules are secreted by fibroblasts, the
propeptides procollagen are cleaved by specific proteases called procollagen peptidases,
forming tropocollagen molecules, as shown in fig 2.2. For each type of collagen, there is
one protease for the amino-terminal propeptide and another protease for the carboxyl-
terminal propeptide. This proteolytic cleavage of propeptides is required for the fiber
formation because they prevent the premature formation of the fiber. Defective removal
of propeptides can lead to generalized disorders of connective tissue (Stryer 1988).
Figure 2.2: Schematic diagram of the conversion of procollagen into tropocollagen
by the excision of the amino-and carboxyl terminals modified from
(Stryer 1988)
After the cleavage of propeptides, the tropocollagens are arranged in staggered fashion
according to the Hodge-Petruska scheme to form collagen fibers (Hodge and Petruska
1963). Tropocollagens are 300 nm long, separated in one row by 40 nm gaps and the
adjacent rows are displaced by 67 nm, as shown in fig 2.3. The 40-nm gap between
adjacent tropocollagen molecules in a row is important in enabling collagen to become
cross-linked after the fiber forms. This gap may also play a role in bone formation (Stryer
N-propeptide Procollagen
Procollagen Peptidases
Tropocollagen C-telopeptide
C-propeptide
N-telopeptide
2. State of the Art
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1988). The last step in collagen synthesis is the formation of intramolecular covalent
cross-links, which will be discussed in details in the next section.
Figure 2.3: Schematic representation of the basic structural design of a collagen
fiber, modified from (Stryer 1988).
2.2.3 Collagen cross-links
Although cross-linking occurs extracellularly, the nature of cross-links also depends on
previous intracellular post-translational modifications to the collagen molecule, in
particular hydroxylation of lysine residues (Knott and Bailey 1998). The collagen is
cross-linked by a unique mechanism based on aldehyde formation from lysine or
hydroxyl lysine side chains by the enzyme lysyl oxidase. Lysyl oxidase (EC 1.4.4.13), the
only enzyme required for cross-link formation, converts the amine side chains of specific
lysine and hydroxylysine residues into aldehyde. Inhibition of this enzyme has deep
effects on the strength of bone and many other tissues because of the subsequent
reduction in cross-linking. Two pathways of cross-linking can be defined in the fibrillar
collagens (Eyre 1984), one based on allysine, the lysine-derived aldehyde, the other on
hydroxyallysine, the hydroxylysine-derived aldehyde. The latter pathway is dominant in
most connective tissues except skin. 3-Hydroxypyridinium residues are the adult cross-
links on the hydroxyallysine route. Two forms of 3-hydroxypyridinium have been
Tropocollagen molecule
40-nm gap 300 nm
67 nm
2. State of the Art
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identified, hydroxylysyl pyridinoline, HP, and lysyl pyridinoline, LP. The location of
cross-linking residues are evident in molecules of type I, II and III collagens (Miller
1971; Knott and Bailey 1998). There are four cross-linking sites, one in each of
telopeptide (residues 9N and 16C) and two sites in the helix (residues 930 and 87).
Figure 2.4: The hydroxyallysine route of collagen cross-linking, modified from (Eyre
1984).
The immature hydroxylysine aldehyde derived cross-links
After the formation of hydroxyallysine, hydroxylysine derived aldehyde, the immature
cross-links are derived from hydroxyallysine. These cross links can be identified in vitro
with borohydride reduction technique. Two Schiff bases (aldimines), borohydride
reducible cross-links, are formed from the condensation reaction, followed by bohydride
reduction, of hydroxyallysine with lysine (hydroxylysinonorleucine, HLN) or with
hydroxylysine (dihydroxylysinonorleucine, DHLN), as shown in fig 2.4. These Schiff
bases are unstable and undergo immediate, spontaneous Amadori rearrangement to form
ketoamines, lysino-5-ketonorleucine (LKNL) and hydroxylysino-5-ketonorleucine
(HLKNL). Borohydride reduction of these ketoamines gives also HLN and DHLN,
making their individual quantification difficult (Mechanic, Kuboki et al. 1974). All forms
Hydroxylysine
Lysyl oxidase
Hydroxylysine
∆HLN ∆DHLN
HLKNL LKNL
HP and LP
DHLN HLN
NaBH4
NaBH4NaBH4
NaBH4
+ Hyl+ Lyl
2. State of the Art
16
of reducible cross-link fall in concentration as connective tissues mature (Fujii and
Tanzer 1974).
The mature enzymatic cross-links (Hydroxypyridinium)
Hydroxylysyl pyridinoline, HP, the most abundant form of the mature cross-links found
in a wide variety of tissues, was first discovered by Fujimoto et al. in bovine Achilles
tendon (Fujimoto, Akiba et al. 1977; Fujimoto, Moriguchi et al. 1978). It is composed of
two hydroxyallysine residues and a helical hydroxylysine and predominant in highly
hydroxylated collagens such as type II in cartilage. A less abundant form, Lysyl
pyridinoline (LP) is found primarily in calcified tissues and is thought to form from two
hydroxyallysine residues and a lysine residue (Eyre 1984). Robins and Duncan proposed
that HP is formed from the condensation of an HLKNL cross-link with hydroxyallysine
(Robins and Duncan 1983). LP is thought to occur through the reaction of LKNL with a
hydroxyallysine (Ogawa, Ono et al. 1982).
N+
OH
NH2
O OH
NH2
O OH
O
OHNH2
N+
OH
NH2O
OH
NH2
OOH
NH2
O OH
(a) (b)
Figure 2.5: The mature collagen cross-links Hydroxylysyl pyridinoline, HP (a) and
Lysyl pyridinoline, LP (b)
2.2.4 The role of hydroxyproline in collagen stabilization
Collagen has a unique Gly-X-Y repeating sequence, where X and Y are frequently
proline and hydroxyproline (Hyp) respectively. It is known in the literature that Hyp
brings stabilization for the collagen structure, but the way how Hyp stabilizes collagen is
2. State of the Art
17
still debatable. The post-translational modification of proline in the Y position to Hyp has
been shown to grant significant additional stability (Kielty, Hopkinson et al. 1993). A
number of studies have been performed to investigate the stabilizing role played by Hyp.
Gustavson (Gustavson 1955) suggested that Hyp residues stabilize collagen by
participating in direct interchain hydrogen bonding. Later, the collagen model was
proposed from fiber diffraction data (Rich and Crick 1961) and it became clear that steric
considerations would not permit direct hydrogen bonding between the Hyp hydroxyl
groups and peptide carbonyls in the same chain, between different chains in the same
molecule, or between different molecules (Bella, Brodsky et al. 1995). In the Rich and
Crick II (RCII) triple helix structure (Rich and Crick 1961), only one hydrogen bond is
formed directly between amide groups (Gly:NH ··· O=C:X) for each tripeptide unit. This
leaves two carbonyl groups and any amide groups present in the X or Y positions (when
these residues are not imino acids) available for hydrogen bonding. The inability of the
Hyp hydroxyl to form direct hydrogen bonds with backbone carbonyl groups together
with the examinations supporting specific binding of water molecules to collagen chains
led to the idea that stabilization of the collagen triple helix by Hyp residues took place
through intramolecular water bridges, involving those carbonyl and amide groups which
were not participating in the interchain RCII hydrogen-bonding pattern (Ramachandran,
Bansal et al. 1973; Suzuki, Fraser et al. 1980; Bella, Brodsky et al. 1995).
Holmgren et al. (Holmgren, Taylor et al. 1998) disagreed with the suggestion that Hyp
stabilizes collagen by providing sites for hydrogen bonding of water and challenged this
by demonstrating that substitution of 4(R)-fluoroproline for 4(R)-hydroxyproline in
collagen-like peptides further increases the thermal stability of the triple helices, despite
the fact that 4-(R)-fluoroproline does not provide a site for hydrogen bonding of water.
They concluded that the stability granted by Hyp cannot be explained by the additional
water bridges but resulted from an electron withdrawing inductive effect of the hydroxyl
or fluoro group that favors the trans configuration of the peptide bond required for
formation of the triple helix. These results were consistent with previous observations
that Hyp residues enhanced the thermal stability of collagen-like peptides even in a
completely anhydrous environment (Engel, Chen et al. 1977) and also with X-Ray
diffraction analysis, which ascertained that the structure of proline is affected by the
2. State of the Art
18
inductive effect elicited by the hydroxyl group of Hyp (Panasik, Eberhardt et al. 1994).
Furthermore Engel and Prockop (Engel and Prockop 1998) concluded that the water
bridges in collagen do not contribute significantly to the stability of the structures.
Instead, the water molecules may simply be innocent bystanders.
2.2.5 The thermal stability of collagen
One of the best criteria to study the way how the collagen molecule held in its triple helix
is to examine conditions under which the stabilization breaks down (Miles and Bailey
2004). The thermal denaturation of collagen induces unfolding of triple helix into random
coils by breaking the hydrogen bonds (Privalov 1982); the ability of collagen to resist this
unfolding is an indication of its “healthiness”.
The shrinkage or contraction of collagen fiber is used as a gross metric of collagen
denaturation (Weir 1949; Gustavison 1956; Hoermann and Schlebusch 1971). There are
two classes of shrinkage temperature measurements (Lee, Pereira et al. 1995): (1)
hydrothermal isometric tension (HIT) and (2) isotonic shrinkage temperature. Verzar
(Verzar 1964) borrowed ideas from muscle physiology and introduced in 1960 the
hydrothermal isometric tension (HIT). In HIT test, the sample is held at constant length in
an aqueous bath while increasing its temperature linearly with time. HIT tests measure
changes in the force that are needed to maintain the tissue at its fixed length during
heating; the force increases when the tissue tries to shrink against the fixed constraint
(Humphrey 2003). In the isotonic shrinkage temperature, the sample is held either
unconstrained or under isotonic constraint; simultaneous measurements of temperature,
time and length are recorded.
It was proved that the level of dehydration as well as the mineral content affects the
thermal stability of collagen. Kronick and Cooke (Kronick and Cooke 1996) showed that
increasing the mineral content enhances the thermal stability of bone. They found that the
denaturation temperature for fully mineralized, partially mineralized and demineralized
bone was 155, 113 and 63 °C respectively.
Much work has been done to elucidate the state of water in biological systems. Rochdi et
al (Rochdi, Foucat et al. 1999) have studied the dependence of the enthalpy and
temperature of denaturation of collagen on the water content using differential scanning
calorimetry (DSC). They found that increasing the water content increases the
2. State of the Art
19
denaturation enthalpy and decreases the denaturation temperature. Miles and Ghelashvili
(Miles and Ghelashvili 1999) utilized the equations of the entropy of a polymer in a box
(Doi and Edwards 1986) to examine the effect of solvent concentration on the collagen
denaturation. They proposed that dehydration brings stabilization mainly by reducing the
lateral dimensions of the lattice and consequently the entropy of activation. They
suggested that dehydration resulting in stripping away of water bridges that connected via
hydrogen bonds to the triple helix. This stripping away reduces the enthalpy of
denaturation because fewer hydrogen bond need to be broken. This explains the increase
of denaturation enthalpy with increasing the water content.
2.2.6 Advanced glycation end products (AGEs)
Non-enzymatic glycation is a common posttranslational modification of proteins
(DeGroot 2004). Maillard reaction or non-enzymatic glycation of proteins is initiated by
the reaction of sugars with lysine and arginine residues in proteins, and eventually leads
to the formation of advanced glycation end products (AGEs) such as
(carboxymethyl)lysine (CML), (carboxyethyl)lysine (CEL), and cross-links, such as
pentosidine, methylglyoxal-lysine dimer (MOLD), and threosidine (Verzijl, DeGroot et
al. 2002). It is now apparent that the AGE formation can also be initiated by lipid
peroxidation (Januszewski, Alderson et al. 2003).
AGEs accumulate with increasing age (Verzijl, DeGroot et al. 2000), results in increased
stiffness of the cartilage collagen network and subsequently leads to increased
susceptibility of the collagen network to mechanical failure (brittleness). Thus,
accumulation of AGEs could be a molecular mechanism that causes age to be a major
predisposing factor for the development of Osteoarthritis (OA) (Verzijl, DeGroot et al.
2002).
In addition the in vivo AGEs, the heat-generated AGEs can be formed in common foods
during the spontaneous reactions between reducing sugars and proteins or lipids (Baynes
and Thorpe 2000). These AGEs are formed in the presence of heat much more rapidly
and in greater concentrations than the in vivo AGEs (Vlassara, Cai et al. 2002).
Therefore, eating high temperature cooked foods will increase the AGEs content in the
body and consequently accelerates aging.
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2.3 Bone Bone is a living, dynamic connective tissue, which has been evolved to fulfill two
functions (Einhorn 1996): the provision of mechanical integrity for both locomotion and
protection, and involvement in the metabolic pathways associated with mineral
homeostasis. Bone refers to a family of materials each with a somewhat different
structural motif, but all having in common the basic building block, the mineralized
collagen fibril (Weiner and Wagner 1998). This family of materials also contains other
members, dentin, the material that constitutes the inner layers of teeth; cementum, the
thin layer that binds the roots of teeth to the jaw; and mineralized tendons. The diversity
of structures within this family reflects the fine-tuning or adaptation of the structure to its
function (Weiner and Wagner 1998).
2.3.1 Bone composition
Water, Hydroxyapatite and collagen Type I are the major components of the bone family
of materials (Weiner and Wagner 1998). The solid phase of the bone consists of organic
(30%) and inorganic (70%) part (Felsenberg 2001). Hydroxyapatite constitutes 95 % of
the inorganic part, whereas the rest 5 % consists of Mg, K and Na-chlorides and
fluorides. Collagen Type I constitutes 95 % of the organic part and 5 % is represented by
the non-collagenous proteins. The composition of bone is shown in fig 2.6.
66.5%28.5%
Hydroxyapatite
Non-collagenous proteins
Collagen Type I
Chloride, Fluoride
3.5%
1.5%
Figure 2.6: The composition of bone, modified from (Felsenberg 2001)
2. State of the Art
21
2.3.2 Bone hierarchy
Bone has a very complex hierarchical structure and is optimized to achieve a remarkable
mechanical performance (Fratzl, Gupta et al. 2004). In order to understand the
mechanical properties of bone material, it is important to understand the mechanical
properties of its component phases, and the structural relationship between them at the
various levels of hierarchical structural organization (Weiner and Traub 1992; Landis
1995). The levels of bone hierarchy, shown in fig 2.7, are (Rho, Kuhn-Spearing et al.
1998)
Macrostructure
At the macrostructure level, bone is distinguished into the cortical (or compact) and
cancellous (or trabecular) types. Cortical bone is a dense solid mass, represents nearly
80% of the skeletal mass (Berne and Levy 1993). It provides a strength where bending
would be undesirable and it is predominant in the appendicular skeleton. Trabecular
bone, represents 20% of the skeletal mass, is less dense, more elastic, has a higher
turnover rate, and much more porous than cortical bone.
Microstructure
The building block of cortical bone, osteon, consists of different sheets (lamellae, 3-7
µm) of mineralized collagen fibers, which wrap in concentric layers (3–8 lamellae)
around a central canal to form what is known as an osteon or a Haversian system.
Cancellous bone is made of an interconnecting framework of trabeculae in a number of
combinations, all comprising the following basic cellular structures: rod–rod, rod–plate,
or plate–plate. A trabecular rod is about 50–300 µm in diameter.
2. State of the Art
22
Figure 2.7: Hierarchical structural organization of the bone, modified from (Rho,
Kuhn-Spearing et al. 1998)
Sub-microstructure
Bone lamellae are 3–7 µm thick (Marotti 1993), but the arrangement and orientation of
the substance of a lamella is not well known (Rho, Kuhn-Spearing et al. 1998). There
may be differences in the lamellae encountered in cortical and cancellous bone. The
osteonal lamellae are wrapped around a central canal, and sequential concentric lamellae
have fiber orientations alternating with each other, spiraling around the central canal.
Parallel lamellae are arranged to form the trabeculae, the building block of the trabecular
bone.
Nanostructure and sub-nanostructures
The most prominent structures seen at this Nano-scale are the collagen fibers, surrounded
and infiltrated by mineral. The three main materials at the sub-nanostructures are crystals,
Cancellous bone
Cortical bone
Osteon
Lamella Collagen fiber
Collagen molecule
Collagen fibril
Bone crystals
10-500 µm 3-7 µm
0.5 µm 1 nm
Microstructure
Sub-microstructure
Nanostructure
Sub-nanostructure
2. State of the Art
23
collagens, and non-collagenous organic proteins. The mature crystals are plate-shaped
(Weiner and Traub 1992) found within the discrete spaces within the collagen fibrils,
thereby limiting the possible primary growth of the mineral crystals, and forcing the
crystals to be discrete and discontinuous. The average lengths and widths of the plates are
50×25 nm. Crystal thickness is 2–3 nm (Landis 1995). The primary organic component
of the matrix is Type I collagen. Collagen molecules secreted by osteoblasts self-
assemble into fibrils with a specific tertiary structure having a 67 nm periodicity and
40 nm gaps or holes between the ends of the molecules, as shown in fig. 2.3. Non-
collagenous organic proteins, including phosphoproteins, such as osteopontin,
sialoprotein, osteonectin, and osteocalcin, may function to regulate the size, orientation,
and crystal habit of the mineral deposits. Through chelation of calcium or enzymatic
release of phosphorus from these proteins, they may serve as a reservoir for calcium or
phosphate ions for mineral formation. However, additional studies are needed to
conclusively define their actions and mechanisms (Rho, Kuhn-Spearing et al. 1998).
2.4 Pericardium The pericardium is a fibro-serous dense connective tissue membrane which encloses the
heart, providing both an internally lubricated protective scaffold for the contracting
myocardium and a metabolically active membrane which can greatly influence cardiac
performance by participating in local prostaglandin metabolism (Simionescu and
Kefalides 1991). Structurally the pericardium is composed of an inner, serous (visceral)
membrane that lines the pericardial cavity and an outer, fibrous (parietal) layer composed
of densely packed wavy collagen fibers among which flat, elongated fibroblasts are
scattered. The major protein constituents known to date are type I collagen, a low
molecular weight dermatan sulphate proteoglycan (decorin) and elastin (Simionescu,
Deac et al. 1994).
Bovine pericardium is widely used as a biomaterial in the fields of reconstructive and
replacement surgery (Sacks, Chuong et al. 1994). Despite the advantages of bovine
pericardium, represented by the availability and immunology, a major difficulty lies in its
intrinsic structural and mechanical variability (Crofts and Trowbridge 1989; Lee, Haberer
et al. 1989). The structure variability of the pericardium made the characterization and
2. State of the Art
24
modeling of the mechanical properties difficult. Therefore the mechanical properties of
the bovine pericardium is poorly characterized (Sacks, Chuong et al. 1994).
Bovine pericardium is generally considered to be mechanically anisotropic (Lee,
Courtman et al. 1984; Zioupos and Barbenel 1994). Small angle light scattering (SALS)
has been used to quantify the collagen fiber architecture of bovine pericardium (Sacks,
Chuong et al. 1994; Mirnajafi, Raymer et al. 2005). SALS maps indicated large animal-
to-animal variability in fiber architecture (Hiester and Sacks 1998; Hiester and Sacks
1998), precluding the use of an anatomic location as a simple guideline for selecting
structurally consistent specimens. Previous studies on the relation between bovine
pericardium biaxial mechanical properties and tissue structure suggested that the local
collagen fiber architecture plays a major role in determining the degree of mechanical
anisotropy (Sacks, Chuong et al. 1994). This suggests the necessity to select structurally
uniform specimens to avoid the mechanical variability (Sacks and Chuong 1998).
Sacks and Chuong (Sacks and Chuong 1998) suggested a procedure for the selection of
specimens, in which first large sections were scanned to select almost uniform regions, as
shown in fig 2.8. The selection process was aided by a simultaneous display of the
regional averaged preferred fiber directions and orientation index (OI), which facilitated a
quantitative assessment of the uniformity of the collagen fiber architecture. Regions of
good structural consistency were removed, and then rescanned to select smaller and more
uniform regions for the mechanical test.
2. State of the Art
25
Figure 2.8: Overview of the pericardium sorting procedure. (a) A course SALS scan
of an anterior section of the pericardium sac showing where the 50 mm
× 75 mm rectangular cutouts regions were extracted, (b) a rescan of the
cutout showing where the 25 mm × 25 mm biaxial test specimen was
selected, and (c) high spatial resolution scan of the biaxial test specimen
overlaid on gray scale OI values demonstrating high uniformity of both
fiber preferred directions and OI, along with definition of the PD and
XD axes (Sacks and Chuong 1998).
3. The Objectives
26
3 The Objectives The demand for the biological implants is rising continuously due the necessity to use
these implants in many surgical and clinical applications. In order to fulfil the increasing
market demand, the production capacity must be increased in the near future. These facts
show how urgent should the Tutoplat process be intensified or optimized without
affecting the quality of the processed tissues.
In order to intensify or optimize the process, the influence of the whole process as well as
of each step of the process on the structural stability of the processed tissues has to be
examined and analysed carefully. This analysis must be the basis of future optimization
trials.
First the influence of Tutoplast process on the thermal stability of processed tissues has to
be studied because the thermal stability is a good indication of the ‘healthiness’ of the
tissues. Furthermore examining the thermal stability is important to understand the effect
behind the thermal treatment of tissues during some surgical operations. Native and
processed samples are exposed to thermal treatment in furnace, followed by a selective
enzymatic digestion method for the measurements of denatured collagen (DC). Analysis
of the isotonic shrinkage temperature (thermoelasticity) as well as SDS-PAGE is used
also to assess the influence of the Tutoplast-process on the thermal stability of the
processed tissues.
After investigating the influence of the whole process, the influence of each step has to be
examined to check its contribution in the probable modification caused by the process.
For this purpose, native samples were exposed to NaOH, H2O2 and acetone treatment
separately with different variations in the treatment concentration and duration. The
investigation of the influence of each step is followed by measurements of the isotonic
shrinkage temperature, measurements of DC and SDS-PAGE.
The effect of the Tutoplast-process on the mechanical properties of the processed tissues
has to be also investigated using tensile and compression tests.
4. Materials and Methods
27
4 Materials and Methods 4.1 Materials For the execution of the experiments, different bovine materials have been used:
4.1.1 Bovine Bones
Femoral head cancellous bone samples, provided from Tutogen Medical GmbH, were
taken from Norwegian sources in the age of 18-25 months. To avoid the risk of the
bovine spongiform encephalopathy (BSE), the scientific steering committee (SCC)
assessed the geographical BSE risk (GBR) of different countries and came to the
conclusion that it is highly unlikely that BSE could be present in Norway (report on the
assessment of the geographical BSE risk of Norway, July 2000). Therefore the samples
used in the experiments were taken exclusively from Norway.
The bone samples were either kept native or processed according to the Tutoplast®
process, described in section 2.1.3.1. For the mechanical analysis, cubes 1×1×1 cm were
sawed from the native bones and stored in 26% NaCl solution at 8 °C.
The Tutoplast-processed bones were pulverized under liquid nitrogen with a ball mill, as
will be discussed in section 4.2.1.1, sieved and the fractions (< 250 µm), shown in fig 4.1,
were stored at -20 °C for the further experiments and examinations.
Figure 4.1: Tutoplast-processed bovine cancellous bone powder (< 250 µm)
4. Materials and Methods
28
4.1.2 Bovine Pericardium
Native bovine pericardium specimens were taken from Norwegian sources in the age of
18-25 months, mechanically defatted and then either kept as native or processed
according to the Tutoplast® process or lyophilized, as shown in fig 4.2.
The native pericardium was stored in 26% NaCl solution at 8 °C, whereas the Tutoplast-
processed pericardium was stored at room temperature. Furthermore a native pericardium
was washed with distilled water to remove the traces of NaCl, lyophilized for 48 h in a
Christ Alpha 1–4 freeze-drying system (Christ, Osterode am Harz, Germany) and then
stored at room temperature.
(a) (b)
(c)
Figure 4.2: Bovine pericardium (a) native, (b) Tutoplast-processed, (c) native
lyophilized
4. Materials and Methods
29
4.2 Methods For the assessment of the quality of the Xenografts, different quality assurance tests have
been used.
4.2.1 Preparation Steps
For the analysis of bone samples different preparation steps were necessary before the
analysis, whereas no preparation step was done for the analysis of the pericardium
samples.
4.2.1.1 The pulverization of the bones
Demineralization is necessary for the analysis of the bone collagen, especially during the
enzymatic digestion to allow better diffusion of the enzyme. The demineralization of
bone is a slow process which takes several weeks. For the acceleration of the
demineralization, the bones can be pulverized to enhance the diffusion of the
demineralizing solutions and consequently to guarantee faster demineralization of the
bones.
Femoral head cancellous bones were pulverized using three types of mills; milling
machine, micro-dismembrator and ball mill.
The milling machine
The milling machine FP1 (Deckel, Bielefeld, Germany) was used to pulverize the bone
samples. It was possible to pulverize relatively large specimens (in the range of 5×2× 1
cm3) in relatively short time (less than 1 minute). During the grinding process, liquid
nitrogen was always refilled to avoid thermal stresses resulted from the milling process.
The milling process was executed under the following parameters, 1200 rpm and feed
rate of 75 mm/min.
Micro-dismembrator
Micro-dismembrator (Sartorius, Goettingen, Germany) was used also to pulverize the
bone samples. In contrast to the milling machine, it was only possible to pulverize small
samples (in the range of 5×5× 5 mm3); however it takes relatively long time (10-20 min).
The bone samples, the Teflon-shaking flask as well as the 4 mm-stainless steel balls were
cooled for 10 minutes in liquid nitrogen, afterwards the shaking flask, filled with the bone
specimens and the liquid nitrogen, was closed and attached to the micro-dismembrator
4. Materials and Methods
30
before the grinding starts. The grinding process was performed under the following
conditions: 2000 rpm swing frequency and 16 mm swing amplitude.
The ball mill
The ball mill analysette 3 pro (Fritsch, Idar-Oberstein, Germany) was used to pulverize
the bone samples. Relatively thin samples (5 mm), regardless of the length and width,
could be pulverized within relatively short time (2-3 min). The pulverization takes place
with 50 mm stainless steel ball under continuous filling of liquid nitrogen.
For the evaluation of the effect of milling process on the bone structure, especially
collagen, samples taken fresh after the milling as well as after 1-week storage at 8 °C,
were used for the measurements of denatured collagen fraction (DC), as shown in table 4-
1.
Table 4-1: The number of bone powder samples taken from the different mills for
the analysis of DC
Number of samples
Mill type Fresh After 1-week storage
Ball mill 11 12
Milling machine 6 6
Micro-dismembrator 6 6
4.2.1.2 The Demineralization of Bones
For efficient diffusion of the enzymatic digestion, the bones have to be demineralized
(Wang, Bank et al. 2001). For this purpose, approximately 1 g of bone powder (<250 µm)
were demineralized using 50 ml 0.5 M EDTA solution (pH 7.4) in a beaker for 72 h with
stirring at room temperature. The beaker was covered with a watch glass.
4.2.2 The Determination of Denatured Collagen (DC)
A selective enzymatic digestion method has been used for the determination of the
fraction of denatured collagen (DC) for the bone and pericardium samples. It consists of
the following steps:
4. Materials and Methods
31
4.2.2.1 A Selective Digestion Method
Intact triple helical collagen molecules are highly resistant to proteolytic enzymes,
whereas degraded (unwound) collagen is easily digested. This fact was exploited to
develop a simplified method for the quantification of the amount of degraded collagen in
the collagen network of connective tissues. This method involves selective digestion of
degraded collagen by α-chymotrypsin (Bank, Krikken et al. 1997).
After certain treatment step, either thermally or chemically, the demineralized bone
powder samples (~ 10 mg for each) as well as the pericardium (~ 8 × 8 × 0.6 mm3 for
each) samples were digested in microcentrifuge tubes overnight at 37 °C with 0.5 ml
incubation buffer containing 1 mg α-chymotrypsin/ml. The incubation buffer is made of
0.1 M Tris HCl, pH 7.3, containing the proteinase inhibitors, 1 mM iodoacetamide, 1 mM
EDTA, 10 µg/ml Pepstatin-A. α-chymotrypsin digests the denatured collagen exclusively
without affecting the intact collagen, as shown in fig 4.3.
After the α-chymotrypsin digestion step, the supernatant, including the denatured
collagen, is separated from the residue, including the intact collagen by centrifugation
step. The centrifugation is done with micro-centrifuge Biofuge pico (Heraeus, Hanau,
Germany) with 10000 rpm for 5 min.
The supernatant was removed quantitatively and diluted 1:1 with 12 N HCl, whereas the
residue was immersed in 6 N HCl. The supernatant and residue were hydrolyzed at 121
°C for 20-24 h in a heating block MBT 250 (Kleinfeld, Gehrden, Germany). The
hydrolyzates were dried using vacuum exsiccator (Häberle, Gaggenau, Germany)
supported with vacuum pump and cold trap (Häberle, Gaggenau, Germany).
4. Materials and Methods
32
Figure 4.3: The principle of the α-chymotrypsin selective digestive method, modified
from (Bank, Krikken et al. 1997)
4.2.2.2 Spectrophotometeric Determination of the DC
The spectrophotometric determination was done according to SOP prepared by Dr. C.C.
Clark (Department of Orthopaedic Research, University of Pennsylvania) as a
modification of previous method (Switzer and Summer 1971). The method is based on
the determination of the hydroxyproline content (HYP) taking into consideration that
hydroxyproline constitutes 14% of the collagen (Elgawish, Glomb et al. 1996).
First a calibration line, using stock solution (10 mg/l), was constructed to determine the
amount of hydroxyproline, as shown in table 4-2.
Incubation with α-chymotrypsin at 37 °C for 24 h
fragments in the supernatant
denatured collagen intact collagen
no fragments in the supernatant
4. Materials and Methods
33
Table 4-2: The quantities used to construct the calibration line of hydroxyproline
content
Aliquot from stock µg hydroxyproline Volume H2O
0 µl 0 2.30 ml
100 µl* 1.0 2.20 ml
250 µl* 2.5 2.05 ml
350 µl* 3.5 1.95 ml
500 µl* 5.0 1.80 ml
Regarding the determination of HYP content for the samples, after evaporating the HCl,
1ml water is added to each sample, placed in ultrasonic bath for 1 min to disrupt the
samples and then a stock solution is prepared for each sample.
The samples are diluted with water with the factor (1:500) and placed in capped tubes to
begin with the assay:
Assay
• The assay is started with adding 0.5 ml potassium borate buffer to all samples
• 2 ml freshly prepared 0.2 M Chloramine-T is added. In this step, the
hydroxyproline will be oxidized to pyrrole-2-carboxylic acid (Berg 1982)
• After 25 min, 1.2 ml of 3.6 M sodium thiosulfate is added to stop the oxidation
• 1.5 g KCl is added
• In hood, 2.5 ml toluene is added. Shaking by hand to initially disperse KCl is
required, and then shaking is continued for 4 min in automatic shaker
• Centrifugation for 5 min at 1500 rpm in
• Using Pasteur pipette, the toluene (upper) layer is removed and discarded with
being careful not to disturb lower layer.
• The capped tubes are placed in boiling water bath for 30 min. This step will
convert the pyrrole-2-carboxylic to Pyyrole
• After cooling the samples to room temperature, 2.5 ml toluene is added and
shaken again with hand and automatic shaker
4. Materials and Methods
34
• Centrifugation for 5 min at 1500 rpm in
• Carefully 1.5 ml of the toluene (upper) layer is removed and placed in a tube
• To each 2-ml toluene aliquot, 0.6 ml Ehrlich’s reagent is added. The Pyyrole
forms with Ehrlich reagent a chromophore
• After 30 min, the absorbance at 560 nm is read
The content of hydroxyproline is measured in the residue and supernatant and converted
to collagen content. Relating the amount of collagen in the supernatant to the amount in
the residue leads to the determination of DC.
4.2.3 The Measurements of the Extent of Browning
5 samples (8×6 mm2) were taken from native, Tutoplast processed, rehydrated Tutoplast
processed and lyophilized pericardium, heated for 1 h at 185, 200 °C in addition to
unheated control samples and then hydrolyzed in 6 N HCl for 24 h. The extent of
browning was detected spectrophotometrically at 420 nm (Friedman and IMolnar-Per
1990) with Specord 210 (Analytik Jena, Jena, Germany).
4.2.4 The Measurements of the Isotonic Shrinkage temperature
For the evaluation of the quality of the bovine pericardium, the technique of the isotonic
shrinkage has been used, as shown in fig 4.4. Pericardium strips (4×1.5 cm) were strained
between two holders in a vessel of water, one holder is fixed and the other is movable
attached with isotonic load. Heating of the aqueous phase is accomplished with a Bunsen
burner with an approximately average heating rate, 2.5 °C/min. The change in the
temperature and specimen length are simultaneously recorded using PT100 (labfacility,
Teddington, UK) and incremental optical encoder (Agilent Technologies, Santa Clara,
CA, USA) respectively. The encoder contains a single lighting emitting diode (LED) as a
light source. Opposite the emitter is the integrated detector circuit. By shrinkage or
change in length, the code wheel rotates between the emitter and the detector, causing the
light beam to be interrupted by the pattern of spaces and bars on the code wheel. The
analysis and representation of the data is done by program developed on Testpoint
software based on the simultaneous acquisition of the experimental data and the graphical
representation.
4. Materials and Methods
35
4cmφ =
Figure 4.4: Schematic setup of the isotonic shrinkage technique
4.2.5 SDS-PAGE
SDS-PAGE is a technique used in biochemistry, genetics and molecular biology to
separate proteins according to their molecular size. The analysis consists of the following
steps:
Preparation of polyacrylamide Gels (PAA)
Previous examinations (Schwarz 2006) have shown that 15% acrylamide gel with silver
stain staining was the best variant for the current objectives.
Two glass plates with two spacers are used to make a single cassette (Peqlab,
Biotechnologie GmbH, Erlangen Germany). Regardless of the system, preparation
requires casting two different layers of acrylamide between the glass plates. The lower
layer is separating gel and the upper layer is stacking gel.
First the separating gel was prepared, all the constituents of the fine-pored gel (pH 8.2)
were mixed, except N, N, N’, N’-tetramethylethylenediamine (TEMED) and ammonium
persulfate (APS). Thereafter, TEMED and APS are added to the mixture, as shown in
table 4-3.
4. Materials and Methods
36
Table 4-3: The composition of 15% PAA separation gel
Substances/gel volume 10 ml 15 ml
H2Odeionized 2.3 3.4
30% bisacrylamide 5.0 7.5
1.5 M Tris (pH 8.8) 2.5 3.8
10 % SDS 0.1 0.15
10 % APS 0.1 0.15
TEMED 0.004 0.006
The gel must be mixed quickly, poured, overlaid with 1 ml distilled water and allowed to
polymerize for 25-30 min. The water has to be decanted after the polymerization.
The next step is to prepare the stacking gel, during the preparation of stacking gel; all the
constituents are added in the order used in the preparation of the separation gel, as shown
in table 4-4.
Table 4-4: The composition of 5% PAA stacking gel
Substances/gel volume 2 ml 3 ml
H2Odeionized 1.4 2.1
30% bisacrylamide 0.33 0.5
1.5 M Tris (pH 6.8) 0.25 0.38
10 % SDS 0.02 0.03
10 % APS 0.02 0.03
TEMED 0.002 0.003
After the addition of TEMED and APS, the stacking gel is poured on the separating gel,
followed by insertion of combs. The protein lysates are passed through the large-pored
stacking gel (pH 6.8), and gathered at the boarders with the separating gel and
consequently penetrate into the separating gel.
4. Materials and Methods
37
Preparation of the Samples
In order to separate the fragments of the collagen, the samples have to be mixed with a
sample buffer with the volume ratio (1:4). The composition of sample buffer is shown in
table 4-5
Table 4-5: The composition of sample buffer
Substances Quantities required in 10 ml water
Tris (pH 6.8) 0.38 g
SDS 1 g
glycerol 2.5 ml
D,L-dithiothreitol (DTT) 0.38 g
Bromphenol blue 5.0 mg
The objectives of sample preparation are to put the proteins into a denaturing buffer,
rendering them suitable for electrophoresis, and to adjust the concentrations of sample so
that an appropriate amount of protein can be loaded onto a gel.
Assembling, Loading, and Running Gels
The assembly of a gel running stand varies with the type of apparatus. The top of the
cassette must be continuous with an upper buffer chamber and the bottom must be
continuous with a lower chamber so that current will run through the gel itself.
The samples, including standard Mark 12® (Invitrogen, Karlsruhe, Germany) and α-
chymotrypsin as reference, are loaded. The anode must be connected to the bottom
chamber and the cathode to the top chamber. The negatively-charged proteins will move
toward the anode. Gels are usually run at a voltage that will run the tracking dye to the
bottom as quickly as possible without overheating the gels, in this case 300 V.
Disassembly and staining
When the dye front is nearly at the bottom of the gel, it is the time to stop the run. The
plates are separated and the gels are washed shortly with distilled water. Subsequently the
4. Materials and Methods
38
gels are incubated in fixation solution overnight at 4 °C. The composition of the fixation
solution is shown in table 4-6.
Table 4-6: the composition of the fixation solution
Substances Quantities (µl)
Ethanol 100 % 100
Acetic acid 20
H2Odeionized 80
Formalin 37 % 0.1
After the incubation, the gels are washed twice with a washing solution (50 % ethanol)
for 25 min. the gels are then stained using silver stain for 20 min; the composition is
shown in table 4-7.
Table 4-7: The composition of silver stain
Substances Quantities
AgNO3 0.2 g
H2Odeionized 100 ml
Formalin 37% 75 µl
After that they are washed with distilled water and incubated in developer solution for 3-
5 min until the bands become visible. Finally the gels are laid in stop solution for 10 min
until the end of the development reaction.
Documentation
The gels are laid on converter screen plate, illuminated with white light; black-white
photo was taken and finished with the help of the program Vision Capt (Peqlab,
Biotechnologie GmbH, Erlangen Germany) (Koerber 2006).
4. Materials and Methods
39
4.2.6 Characterization of the Mechanical Properties
The mechanical testing machine Instron model 5569 (Instron Ltd, High Wycombe, UK)
in association with Instron Merlin software has been used for the characterization of the
mechanical properties of the bone and pericardium at room temperature.
The bones are tested using the compression test, first the sample was placed on the
fastening plate beneath the load cell. The load cell starts to descend and compress the
sample. The compression proceeds and the force acting on the sample increases until it
reaches the maximum value then it decreases. The test can be stopped when the force
reaches a pre-adjusted value below the maximum, e.g. 50%. The data is analyzed using
the Merlin software to construct the required stress-strain diagram.
The temperature dependent mechanical properties of the bone were examined. For this
purpose, 9 (1×1×1 cm3) samples were taken and heated for 1 h at each temperature of the
following temperatures 37, 60, 80 and 100 °C, before being mechanically tested.
The pericardium sample (8×1.5 cm2 strip) is tested using the tensile test, in which the
load cell used in the compression test is removed. Alternatively, the sample is inserted
between two clamps, upper and lower. The upper clamp starts to pull the sample causing
extension of the sample. The test is stopped when the sample is extended to pre-adjusted
value, e.g. 200%. The data is analyzed using the Merlin software to construct the required
stress-strain diagram.
The influence of Tutoplast processing on the mechanical properties of the pericardium
was examined. For this purpose, 10 (8×1.5 cm2) strips were cut from the left side of the
pericardium sac and 10 strips from the right side. From each side 5 strips were processed
and the other 5 kept unprocessed as control.
4.2.7 The measurements of the Thermal Conductivity
5 (8×6 mm2) native as well as 5 processed samples were submerged in 30 ml distilled
water for 1 h, thereafter the conductivity of the water was measured using conductivity
meter WTW LF95 (WTW, Weilheim, Germany).
4.3 Different physical and chemical treatment During the current work the following chemical and thermal treatments have been
executed:
4. Materials and Methods
40
4.3.1 The thermal treatment of collagenous tissues
4.3.1.1 The thermal treatment of bovine bone
Bovine cancellous bone samples, taken from Norwegian sources in the age of 18-25
months, were processed according to Tutoplast process in January 2005, pulverized with
milling machine under liquid nitrogen and the fraction (< 250 µm) was stored at -20 °C in
March 2005. The thermal stability of bone powder was examined during April and May
2005 and also September and October 2005.
The bone powder samples were heated for 1 h at each temperature in the range between
(55-200 °C), as shown in table 4-8.
Table 4-8: The samples used for the thermal treatment of bovine bone
Temperature
(°C)
25 55 90 120 135 150 160 170 200
No. of
samples
6 5 6 9 9 9 9 9 6
For temperatures lower than 100 °C; the samples were heated in a water bath, whereas at
temperatures higher than 100 °C, the samples were incubated for 15 min in boiling water
bath and then heated in a furnace to the corresponding temperature. Afterwards, the
samples were demineralized with 0.5 M EDTA (pH 7.4) for three days, incubated with α-
chymotrypsin overnight at 37 °C for the determination of DC.
4.3.1.2 The thermal stability of bovine pericardium
For the evaluation of the thermal stability of bovine pericardium, bovine pericardium
specimens from Norwegian sources in the age of 18-25 months were stored under 26 %
NaCl solution at 8 °C in January 2006. The thermal stability of the native and Tutoplast-
processed pericardium sample was examined during February and March 2006, whereas
the thermal stability of lyophilized pericardium during August and September 2006.
4. Materials and Methods
41
For the measurements of DC
For the evaluation of the thermal stability of native bovine pericardium, 5 samples (8×6
mm2) were heated at each temperature of the following temperatures (25, 55, 70, 80, 90,
100, 110, 120, 135, 150, 160, 170, 185 and 200 ° C) for 1 h in furnace.
For the evaluation of the thermal stability of Tutoplast-processed bovine pericardium, a
part of a native pericardium was processed with Tutoplast process. 5 samples (8×6 mm2)
were heated at each temperature of the following temperatures (25, 55, 70, 80, 90, 100,
110, 120, 135, 150, 160, 170, 185 and 200 ° C) for 1 h in furnace.
For the evaluation of the thermal stability of the native lyophilized bovine pericardium, a
part of a native pericardium was lyophilized for 48 h in a Christ Alpha 1–4 freeze-drying
system (Christ, Osterode am Harz, Germany). 5 samples (8×6 mm2) were heated at each
temperature of the following temperatures (25, 55, 70, 80, 90, 100, 110, 120, 135, 150,
160, 170, 185 and 200 ° C) for 1 h in furnace.
For the investigation of the kinetic and thermodynamic parameters, isothermal
experiments have been performed. For this purpose, samples of Tutoplast processed and
lyophilized pericardium were taken and heated for 5, 10, 15 and 20 min in furnace at
temperatures 145, 160 and 175 °C (3 samples for each time at each temperature).
After the thermal treatment the samples were incubated with α-chymotrypsin overnight at
37 °C for the determination of DC.
SDS-PAGE
At each temperature from the following temperatures, 25, 55, 90, 120, 135, 150, 160, 170
and 185 °C, 2 (8×6 mm2) native and 2 Tutoplast-processed pericardium samples were
heated for 1 h. After the thermal treatment, the samples were incubated with α-
chymotrypsin overnight at 37 °C. The supernatant, containing the denatured collagen
part, was taken for SDS-PAGE investigations.
4.3.2 The Sodium hydroxide treatment and the corresponding neutralization
4.3.2.1 Sodium hydroxide treatment
For the evaluation of the sodium hydroxide treatment, bovine pericardium specimens
from Norwegian sources in the age of 18-25 months were stored under 26 % NaCl
solution at 8 °C in May 2006.
4. Materials and Methods
42
Measurement of the extent of hydrolysis
For the measurement of the extent of hydrolysis at room temperature, 10 native and 10
Tutoplast-processed (8×6 mm2) pericardium samples, with average dry weight of 16.8
and 12 mg respectively, were treated with 1 N NaOH for different durations (0, 30, 60,
90, 120 and 150 min) in August 2006. The extent of hydrolysis was measured
spectrophotometrically at 280 nm based on the content of the aromatic amino acids. For
this purpose, a calibration line for the content of aromatic amino acids was constructed
using a stock solution of 2 mg aromatic amino acids in 100 ml water. 5 samples contain
the following weights (0, 2.5, 5, 7.5, 10, 12.5, 15, 20 µg aromatic amino acids) were used
to construct the calibration line (Koerber 2006).
Shrinkage temperature measurements
5 Strips (4×1.5 cm) were cut from a native pericardium in June 2006 and treated with 50
ml 1 N NaOH for 1 h, shortly washed with water and immersed in 0.9% NaCl solution
for 24 h before being tested. 5 control untreated strip were also tested.
Measurements of DC
5 (8×6 mm2) native pericardium samples were treated with 40 ml 1 N NaOH for 1 h and
incubated with α-chymotrypsin for the determination of DC. 5 control untreated samples
were also tested
SDS-PAGE
At each concentration and each time from the following 0.5 N (30 min, 1 h, 2 h), 1.0 N
(30 min, 1 h, 2 h) and 2.0 N (30 min, 1 h, 2 (8×6 mm2) native pericardium samples were
treated with NaOH. After the treatment, the samples were incubated with α-chymotrypsin
overnight at 37 °C. The supernatant, containing the denatured collagen part, was taken for
SDS-PAGE investigations.
4.3.2.2 The neutralization of the tissues after the NaOH treatment
In order to neutralize the pericardium samples after the NaOH treatment, 10 native
pericardium strips (4×1.5 cm) were treated with 100 ml 1 N NaOH for 1 h, 5 samples of
them were treated with 50 ml 1 N CH3COOH for 5 min and the other 5 samples with 50
ml 1 N CH3COOH for 15 min. This treatment was performed in June 2006.
For the examination of the influence of the concentration CH3COOH, 10 native strips
(4×1.5 cm) were treated with 100 ml 1 N NaOH for 1 h, 5 of them neutralized with 50 ml
4. Materials and Methods
43
1 N CH3COOH for 15 min and the other 5 with 50 ml 0.1 N CH3COOH for 15 min. This
treatment was performed in November 2006.
For the evaluation of the efficiency of washing solutions after the NaOH treatment, 15
native strips (4×1.5 cm) were treated for 1 h with 150 ml NaOH, 5 of them were
immersed for 30 min in 50 ml distilled water, another 5 were washed under stirring for 30
min with 50 ml 100 mmol phosphate buffer (pH 7.4) and the other 5 washed under
stirring for 90 min in 50 ml distilled water, in which water was changed every 15 min.
This treatment was performed in September 2006.
For the evaluation of efficiency of acid treatment step followed by washing step after the
NaOH treatment, 10 strips were treated for 1 h with 100 ml 1 N NaOH and treated with
100 ml 0.1 N CH3COOH for 15 min, subsequently 5 of them were washed under stirring
twice for 10 min with 50 ml 100 mmol phosphate buffer and the other 5 with 50 ml
distilled water. This treatment was performed in November 2006.
4.3.3 The hydrogen peroxide treatment
Shrinkage temperature measurements
12 native bovine pericardium strips (4×1.5 cm) were treated with H2O2 for 48 h, 4 of
them with 3 %, another 4 with 10 %, and the other 4 with 30 % H2O2, immersed in 0.9 %
NaCl solution for 24 h before being tested with the isotonic shrinkage technique in June
2006.
Measurements of DC
15 (8×6 mm2) native pericardium samples were treated H2O2 for 48 h, 5 of them with 3
%, another 5 with 10 %, and the other 5 with 30 % H2O2 and incubated with α-
chymotrypsin for the determination of DC. 5 control untreated samples were also tested
4.3.4 The acetone treatment
For the evaluation of the acetone treatment, bovine pericardium specimens from
Norwegian sources in the age of 18-25 months were stored under 26 % NaCl solution at 8
°C in April 2006.
The extent of drying and volume shrinkage
Two native bovine pericardium groups were exposed to acetone treatment in May 2006
for 18 days, the first group with 100% acetone and the second with acetone series starting
from 30% until 100%, every two days increased by 10% (Koerber 2006). Each 2 days, 5
4. Materials and Methods
44
samples 24×26×1.0 mm3 were taken for the measurements of the extent of drying and 1
sample for the volume shrinkage measurements from both groups (pure acetone and the
acetone series)
The measurements of DC
9 native bovine pericardium samples were subjected to 100% acetone and 6 to acetone
series treatment for 18 days, in addition to 10 samples untreated, incubated overnight
with α-chymotrypsin for the determination of DC (Koerber 2006).
4.3.5 The determination of water content
For the determination of water content, samples taken from bovine bone, native
pericardium, Tutoplast-processed pericardium, and lyophilized pericardium and heated
for 1 h at 105 °C, as shown in table 4-9. The weight loss was considered as the water
content
Table 4-9: The samples used in determining the water content
Substance Native
pericardium
(8×8 mm2)
Tutoplast-
processed
pericardium
(8×8 mm2)
Lyophilized
pericardium
(8×8 mm2)
Bone powder
No. of samples 10 10 10 5
5. Results Interpretations
45
5 Results Interpretations 5.1 The effect of bone pulverization on the collagen As described in section 4.2.1.1, the digestion of bone samples with α-chymotrypin
requires bone demineralization to guarantee the diffusion of α-chymotrypin (Wang, Bank
et al. 2001). In order to accelerate the bone demineralization, the bone samples were
pulverized; three types of mills have been used to pulverize femoral head cancellous
bones, milling machine, micro-dismembrator and ball mill. Subsequently, the powder
was sieved and the fraction (< 250 µm) was taken for the further examinations.
5.1.1 The measurements of DC
The analysis of bone powder using the measurements of denatured collagen (DC) shows
that pulverizing the bones either with the milling machine or with the micro-
dismembrator is destructive represented by the high values of DC, they have DC, mean ±
95% confidence interval, 56.6 % ± 6.2 and 68.4 % ± 20.7 respectively. In contrast to the
milling machine and the micro-dismembrator, ball mill is significantly the least
destructive mill and has relatively low DC values, 16.6 % ± 4.6. Interestingly the
destruction or denaturation caused by the milling machine and the micro-dismembrator is
temporary. It is confirmed that storing the bone powder for 1 week at 8 °C yields
significantly lower values of DC, 34.1 % ± 5.0 and 27. 0 % ± 6.7 respectively, as seen in
fig 5.1. The DC for the samples pulverized with the ball mill is almost unaffected by the
1 week-storage, 17.9 % ± 2.9.
5. Results Interpretations
46
DC
(%)
0
20
40
60
80
100
ball mill
micro-dismembrator
milling machine
fresh after 1 week Figure 5.1: Investigation of the effect of bone pulverization on the DC (error bars
indicate 95 % confidence interval for 6-12 samples)
5.1.2 Discussion of the results
It is reasonable to test the influence of bone pulverization on the stability of bone and
collagen for two reasons: first, to avoid any interfering influences on further experimental
results done with the bone powder, second because grinding is one the processing steps
for powder and granulate biological implants. It is known that the mechanical grinding or
pulverization can lead to changes in the structure of collagen and to mechanical
denaturation (Segalova, Dubinskaya et al. 1981). Day (Day 2005) has observed that
micro-dismembrator doubled the DC during the pulverization of osteaoarthritic bones. In
the present work pulverization has been done under liquid nitrogen to avoid thermal or
mechanical stress resulted during the process, which could destruct or denature the
collagen molecules in bone. However it was not enough to prevent the denaturation of
bones pulverized with the milling machine as well as with the dismembrator.
Surprisingly, a kind of ‘renaturation’ has been observed after storing the bone powder for
1 week at 8 °C. It is expected that the relatively short time of pulverization can create
5. Results Interpretations
47
moderate heating that results in a local unfolding within the protein, which appears to
regain its native structure upon the restoration of normal temperatures. This unfolding
may be due to the breaking of a small number of consecutive hydrogen bonds (Wright
and Humphrey 2002), in this case, the triple helix is unfolded but the polypeptide chains
are still fixed in their positions (Hoermann and Schlebusch 1971), which enable the
recovery of the native triple helix by building hydrogen bonds.
5.2 The thermal stability of collagenous tissues An excellent mean of studying the way in which the collagen molecule is held in its triple
helix is to examine the conditions under which the stabilization breaks down, helix coil
transition (Miles and Bailey 2004). The thermal denaturation of collagen induces
unfolding of the triple helix into random coils by breaking the hydrogen bonds (Privalov
1982); the ability of collagen to resist this unfolding is an indication of its “healthiness”.
Many imaging techniques such as, micro-computed tomography (Laib, Barou et al. 2000;
Bagi, Hanson et al. 2006) and transmission electron microscope (Porter, Nalla et al. 2005;
Sahar, Hong et al. 2005) can detect structural modification or destruction in µm or nm
scale. In contrast to these techniques the thermal stability covers higher level of
hierarchy.
5.2.1 Analysis of the thermal stability with the measurements of DC
5.2.1.1 The thermal stability of Tutoplast-processed bovine bone
The collagen denaturation induced by the thermal treatment is obviously shown in the
measurements of DC. It is observed, as seen in fig 5.2, that DC for bone powder remains
unchanged till the temperature 90 °C, and then it starts to increase linearly with
increasing temperature until almost 90 % denaturation is reached at temperature 160 °C.
Thereafter the DC is almost unchanged between 160 and 170 °C and then it decreases
slightly between 170 and 200 °C.
5. Results Interpretations
48
Temperature (°C)
0 20 40 60 80 100 120 140 160 180 200 220
DC
(%)
0
20
40
60
80
100
6 56
9
9
9
99
6
Figure 5.2: The temperature dependency of DC for Tutoplast-processed cancellous
bovine bone powder (error bars indicate 95% confidence interval for 5-
9 samples)
5.2.1.2 The thermal stability of native bovine pericardium
The DC of native pericardium (initial water content ~ 85.7%) is sensitive to the
temperature increase, as shown in fig. 5.3. It remains unchanged until 55 °C and then it
starts to increase progressively with increasing temperature until 150 °C. After that it
reaches a plateau between 150 and 200 °C. A significant reduction of the DC has not
been observed up to 200°C.
5. Results Interpretations
49
Temperature (°C)
0 20 40 60 80 100 120 140 160 180 200 220
DC
(%)
0
20
40
60
80
100
Figure 5.3: The temperature dependency of DC for native bovine pericardium
(error bars indicate 95% confidence interval for 5 samples)
5.2.1.3 The thermal stability of tutoplast-processed bovine pericardium
The Tutoplast-processed pericardium (initial water content ~ 1.9%) is extremely
thermally stable, as shown in fig 5.4. The DC is almost constant until 150 °C and then it
jumps too sharply to reach almost complete denaturation at 160 °C. The DC is almost
unchanged in the temperature range between 160 and 170 °C. One of the most interesting
remarks in these results is the sudden decrease of DC in the range (185-200 °C).
5. Results Interpretations
50
Temperature (°C)
0 20 40 60 80 100 120 140 160 180 200 220
DC
(%)
0
20
40
60
80
100
Figure 5.4: The temperature dependency of DC for Tutoplast-processed bovine
pericardium (error bars indicate 95% confidence interval for 5
samples)
5.2.1.4 The thermal stability of the lyophilized bovine pericardium
As observed with the Tutoplast-processed pericardium, the lyophilized pericardium
(initial water content ~ 7%) is also extremely thermally stable, as shown in fig. 5.5. The
DC is almost unchanged until 135 °C and then it increases but smoothly with increasing
temperature until 170 °C. An extreme reduction of DC in the range (185-200°C) has also
been shown with the lyophilized pericardium.
5. Results Interpretations
51
Temperature (°C)
0 20 40 60 80 100 120 140 160 180 200 220
DC
(%)
0
20
40
60
80
100
Figure 5.5: The temperature dependency of DC for the native lyophilized bovine
pericardium (error bars indicate 95% confidence interval for 5
samples)
5.2.1.5 The measurements of the extent of browning
The analysis of extent of browning, measured at 420 nm, shows that the absorbance of
the lyophilized and Tutoplast processed pericardium is slightly higher than that of the
native and rehydrated Tutoplast processed at 185 °C, as shown in fig. 5.6. The extent of
browning of the lyophilized and Tutoplsat processed pericardium becomes significantly
higher at 200 °C
5. Results Interpretations
52
Abs
orba
nce
(-)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
RT 185 °C 200 °C
rehydrated processed
processed
native lyophilized
native
Figure 5.6: The measurements of the extent of browning at 420 nm for native, native
lyophilized, rehydrated Tutoplast and Tutoplast-processed bovine
pericardium at room temperature, 185 and 200 °C (error bars indicate
± standard deviation for 5 samples)
5.2.1.6 Discussion of the results
As seen in figures 5.2-5.5 and 5.7, there was always an initial value of DC (around 16-22
%) even before the thermal treatment. The detection of denatured collagen in the native
pericardium at the beginning can be probably justified by the presence of soluble
collagen molecules, such as the newly synthesized and the other non-cross-linked
collagen (Bank, Krikken et al. 1997). Furthermore the effect of mechanical defatting and
the cutting of samples can’t be excluded. The initial value of DC for Tutoplast-processed
pericardium is possibly resulted from the processing, whereas contribution of soluble
proteins in this initial value is not expected because such proteins must be eliminated
during the hydrogen peroxide oxidation step in the process (Schoepf 2006). The initial
DC value of the lyophilized pericardium is quite higher than those of the native and the
5. Results Interpretations
53
solvent-processed. It is expected that the lyophilization cleaves some terminal collagen
domains, makes them accessible for α-chymotrypsin.
The denaturation or melting temperature (at DC = 0.5) is an important parameter to
assess the thermal stability of collagen. According to the results of the thermal stability of
Tutoplast-processed bovine cancellous bone, the denaturation temperature is
approximately 135 °C, as shown in fig 5.2, which differs from values got by other
research groups. The degree of mineralization (Kronick and Cooke 1996) as well as the
water content (Miles and Ghelashvili 1999) could cause this difference in denaturation
temperature. Kronick et al (Kronick and Cooke 1996) proved the influence of the degree
of mineralization on the denaturation temperature of collagen using differential scanning
calorimetry (DSC) measurements, they observed a denaturation temperature of 155 °C
for fully mineralized bone, while 113 °C and 63 °C for partially mineralized and
demineralized bone respectively. However, they didn’t mention the absolute mineral
content of the samples. Fratzl et al (Fratzl, Gupta et al. 2004) suggested that bone is not
uniformly mineralized but composed of bone packets, each having its own mineral
content. Therefore the sampling site could also affect the denaturation temperature. In the
current work, the bones are pulverized to avoid the effect of the heterogeneity in mineral
distribution; however the mineral content of the samples has not been determined.
Regarding the water content, Trebacz and Wojtoowicz (Trebacz and Wojtowicz 2005)
have tested the influence of water content on the denaturation temperature of cortical and
trabecular bone samples using DSC and observed for samples of water content between
0-8% a hydration-dependent peak within a range from 154 to 161 C. Wang et al (Wang,
Bank et al. 2001) observed a denaturation temperature of 160 °C for cortical bone
samples after selective digestion with α-Chymotrypsin without statement about the water
content. In this work, the powder samples are heated in boiling water bath for the
temperatures lower than 100 °C. In order to have relatively similar conditions for all the
samples regarding the effect of moisture content on the thermal stability, the samples for
the temperatures above 100 °C are heated for 15 min in boiling water bath before being
heated in furnace. This preheating step in water bath increases the water content of the
samples to approximately 59%.
5. Results Interpretations
54
Concerning the thermal stability of bovine pericardium, The DC curve of native,
Tutoplast-processed and lyophilized pericardium shows a similar sigmoidal behavior.
The curve consists of 3 regions; the length of each region is different from one tissue to
another depending on the water content and the preprocessing. The initial region of the
curve, where almost no change is observed, is followed by a region, where DC
approximately increases linearly with temperature. The final region is represented by
plateau or decrease of DC depending on the preprocessing.
The DC of the native pericardium (initial water content ~ 85.7%) shows higher sensitivity
to temperature change than that of the Tutoplast-processed (initial water content ~ 1.9%)
and the lyophilized (initial water content ~ 7%). It starts to increase linearly from 55 °C
until it reaches a plateau at 150 °C. The denaturation temperature (at DC = 0.5) is
approximately 115 °C, which differs from the reported values of shrinkage temperature
of native bovine pericardium in the literature (60-65 °C) (Pasquino, Pascale et al. 1994;
Moore, Chen et al. 1996). The measurements of shrinkage temperature as indication of
collagen denaturation are performed under fully hydrated conditions in water bath,
whereas the measurements of DC are taken after thermal treatment in furnace, in which
the thermal stability behaves differently, as will discussed later in the polymer in a box
mechanism.
Over a temperature range from 150 to 200 °C the DC is temperature independent. The
logical explanation for this behaviour is that the thermal denaturation of collagen is
kinetically controlled. A same level of heating damage can be obtained by combinations
of heating time and temperature level (Wright and Humphrey 2002). It is expected that
the 1-h heating was too long to examine the effect of increasing temperature on the DC,
in other words, with this relatively long time, the maximum limit of damage can be
reached in this range regardless of the temperature.
5. Results Interpretations
55
Temperature (°C)
0 20 40 60 80 100 120 140 160 180 200 220
DC
(%)
0
20
40
60
80
100
native
native lyophilized
processed
Figure 5.7: The temperature dependency of DC for native, Tutoplast-processed and
native lyophilized bovine pericardium (error bars indicate 95%
confidence interval for 5 samples)
A possible reason for not reaching complete denaturation or complete enzymatic
digestion at high temperatures could be the presence of proteoglycans, which prevent α-
chymotrypsin to diffuse easily (Bank, Krikken et al. 1997).
In contrast to the native pericardium, the Tutoplast-processed and lyophilized
pericardiums are extremely thermally stable. A relatively large initial region, where DC is
almost unchanged, is ended by a sharp denaturation. A final region, characterized by the
reduction of DC, has been observed in both cases.
The Tutoplast-processed pericardium is thermally slightly more stable than the
lyophilized one; the DC for Tutoplast-processed samples remained constant until
approximately 150 °C, whereas for the lyophilized samples, it was almost unchanged
until 135 °C. Dehydration in the lyophilized and Tutoplast-processed pericardium
5. Results Interpretations
56
obviously gives the samples a kind of protection against thermal denaturation. According
to the polymer in a box mechanism (Doi and Edwards 1986; Miles and Ghelashvili 1999)
dehydration reduces the lateral dimensions of the lattice, constrains the number of
possible configurations, reduces the free-volume available for denaturing α-chains
(Trebacz and Wojtowicz 2005), reduces the configuration entropy and thereby increases
the thermal stability of collagen. The slight preference for the Tutoplast-processed against
the lyophilized pericardium could be attributed to the lower water content of the
Tutoplast-processed pericardium. The dehydration constrains the movement of molecules
to be denatured and appeared to be confined within a “box”, as shown in fig 5.8. It is
supposed that the less the water content is, the more the collagen within this box is
confined, i.e. the Tutoplast-processed is more confined and consequently thermally more
stable.
Figure 5.8: schematic illustration shows the therma activation of collagen molecules
under higher and lower water content according to polymer in a box
mechanism, modified from (Miles and Ghelashvili 1999)
According to polymer in a box mechanism, the water content determine the size of the
box, the higher the water content, the larger the box. Therefore, the collagen molecules in
Low water content
Thermal activation
High water content
Thermal activation
5. Results Interpretations
57
the native pericardium are confined within large box, which couldn’t restrict the
movement of molecules to be denatured. During the thermal treatment of the native
pericardium, the molecules begin to denature with simultaneous reduction of water
content due to the evaporation. This reduction of water content leads to reduction of the
dimensions of box confining the molecules. After certain time, depending on the
temperature, the water is totally evaporated and the collagen molecules are confined
within small box. At this time, the denaturation of collagen proceeds with a rate lower
than that at the beginning of the heating but still higher than that of the originally dry
tissues (in this case Tutoplast-processed and lyophilized) because this box constrains
partly denatured or disordered molecules, whereas the box in the case of the dry tissues
contains intact and well-ordered molecules.
Understanding the polymer in a box mechanism is helpful to differentiate between
heating wet samples in furnace and heating wet samples in water bath. During the heating
in furnace, the dimensions of the box become smaller due to the evaporation of water,
whereas no evaporation of water takes place during the heating in water bath and
consequently no reduction of the box dimensions. Therefore the tissues are thermally less
stable when they heated in water bath.
A very remarkable observation was the extremely sharp increase of DC for the Tutoplast-
processed pericardium within 10 degrees (160-170 °C), in comparison with the relatively
smooth increase for that of lyophilized pericardium (135-170 °C). It is thought that
dehydration restricts the movement of collagen molecules and become confined within a
“box”, which protects collagen molecules from thermal denaturation. The molecules are
located within this hypothesized box. In order to denature collagen molecules, the
“protective box” has to be overcome and then the molecules remain unprotected against
thermal denaturation. In the case of Tutoplat-processed pericardium, after the supposed
removal of the protective box, the pericardium, which contains partly modified collagen
molecules, has lower ability to resist the thermal denaturation, which explains the sharp
increase of DC in the range 160-170 °C, as shown in fig 5.7. In contrast to Tutoplast-
processed pericardium, the lyophilized pericardium contains relatively “healthy” and
intact collagen molecules that could resist the thermal denaturation causing smooth not
sharp denaturation in the range 135-170 °C.
5. Results Interpretations
58
It was seen in general that lyophilized and Tutoplast-processed pericardium has sharper
denaturation in comparison with the native pericardium. As the collagen further
dehydrated, water molecules are stripped away causing an increase of the thermally-labile
domain and consequently a sharper denaturation (Miles and Ghelashvili 1999).
One of the most interesting remarks in DC results is the sudden decrease of DC for the
Tutoplast-processed and lyophilized pericardium in the range 185-200 °C. There are two
possible reasons behind the reduction of DC or the inability of α-chymotrypsin to digest
the denatured collagen at high temperatures, the breaking of covalent cross-links or the
formation of heat-generated advanced glycation end products (AGEs). Trebacz and
Wojtowicz (Trebacz and Wojtowicz 2005) have recognized two endothermal peaks
during DSC study of bones and tendons, the first endotherm was in the range from 155 to
165 °C for bones and from 118 to 137 °C for tendons, whereas the second endotherm was
in the range from 245 to 290 °C for bones and from 200 to 285 °C for tendons. They
supposed that that second peak, which accompanied with brown bones, is related to the
breaking of covalent cross-links. In the other hand, Maillard (browning) reaction or non-
enzymatic glycation is initiated in vivo from the reaction of reduced sugar with lysine and
arginine residues in protein (Verzijl, DeGroot et al. 2002). Furthermore AGEs formation
can be initiated by lipid peroxidation (Januszewski, Alderson et al. 2003). In contrast to
the in vivo AGEs, the heat generated AGEs, generated during the spontaneous reaction
between reducing sugars and proteins or lipids, form much rapidly and in greater
concentrations (Vlassara, Cai et al. 2002). In this study, it is expected the formation of
heat-generated AGEs, not the breaking of cross-links, was behind the reduction of DC.
The presence of proteoglycans in the pericardium tissues (Simionescu and Kefalides
1991; Simionescu, Deac et al. 1994) leads to the formation of AGEs at high temperatures,
which hinders the enzymatic degradation of collagen (DeGroot, Verzijl et al. 2001). The
reduction of DC was observed at relatively low temperatures in this study, from 185 °C,
in comparison with the supposed reported temperatures for the breaking cross-links
(Trebacz and Wojtowicz 2005). Furthermore the samples were characterized by a light
brown colour not dark brown as one expected in case of organic decomposition.
Spectrophotometrical measurements of the extent of browning prove that the lyophilized
and Tutoplast-processed pericardium formed heat-generated AGEs in the range (185-200
5. Results Interpretations
59
°C). The absorbance of browning for the lyophilized and Tutoplast-processed was
slightly higher than that of the native and rehydrated Tutoplast-processed at 185 °C and
become significantly higher at 200 °C as indication of the formation of heat generated
AGEs. The reason behind the absence of AGEs with the native pericardium (water
content ~ 85.7 %) as well as rehydrated Tutoplast-processed (water content ~ 94.0 %)
could be ascribed to the higher moisture content, which probably delays the reaction of
AGEs formation (Vlassara, Cai et al. 2002). It is supposed that the thermal denaturation
under high moisture-content proceeds too quickly preventing the formation of AGEs,
whereas under dry condition, the denaturation doesn’t proceeds quickly due the higher
resistance against the thermal denaturation, which allows the formation of heat-generated
AGEs. These results could explain the sudden reduction of DC at higher temperatures
with the Tutoplast-processed and the lyophilized but not with the native pericardium. The
current results confirm that the Proteoglycans are not removed during Tutoplast-process.
The slight not sharp reduction of DC, observed with the bone powder at high
temperature, could be attributed to the moderate water content, 59%.
5.2.1.7 Modeling of the thermal denaturation of pericardium
The thermal stability of collagen is one of the controversial subjects whether the collagen
denaturation is an equilibrium process or kinetic process. The Lumry and Eyring model
was established in 1954 to study the conformation changes in globular proteins, with
intermediate state that could either refold to the original native state or convert
irreversibly to the final denatured state (Lumry and Eyring 1954). The simplest form of
the Lumry and Eyring model can be presented by the following scheme (Sanchez-Ruiz
1992):
FUNkK→⇔ (5.1)
where N , U , and F are the native, unfolded and final (irreversibly denatured) states of
the protein, respectively.
Sanchez-Ruiz assumed that there is a reversible chemical equilibrium between N and U
and the unfolding enthalpy is constant. The van’t Hoff enthalpy H∆ can be obtained
from the temperature dependency of the equilibrium constant between the native and
unfolded states:
5. Results Interpretations
60
2
lnRT
HdT
Kd ∆= (5.2)
where R is the ideal gas constant. Assuming that H∆ is temperature independent, the
equilibrium constant K can be expressed as (Sanchez-Ruiz 1992):
[ ][ ] [ ]
⎭⎬⎫
⎩⎨⎧
−∆−=== mN
U TTRH
XX
NUK /1/1exp (5.3)
where xuand xN
are the molar fractions of unfolded and native states and Tm is the
temperature at which K = 1.0
Sanchez-Ruiz assumed also that the irreversible step ( FU → ) is described by the
kinetics of a first order reaction. The rate equation for the irreversible formation of F is
given in the following equation:
][][ UkdtFd
= , or uF Xk
dtdX = (5.4)
where X F is the molar fraction of the final state
The temperature dependency of the rate constant k, can be expressed with the Arrhenius
equation as:
[ ]⎭⎬⎫
⎩⎨⎧ −−= */1/1exp TT
REk , (5.5)
where E is the activation energy and T* is the temperature at which =k 1.0 min-1
(Sanchez-Ruiz 1992). Taking equation (5.2) into consideration and 1=++ XXX FUN ,
then:
)1(1 XK
kKdt
dXF
F −+
= (5.6)
Equation (5.6) shows that, at constant temperature, X F changes with time after first-order
kinetics with an apparent rate constant equal to 1+K
kK
Privalov (Privalov 1979) developed a theory of the temperature-induced changes of small
globular proteins based on equilibrium thermodynamics. He checked the validity of two-
state model and suggested that if the van’t Hoff enthalpy is identical to the calorimetric
enthalpy, then the denaturation is considered as a two-state process in equilibrium, i.e. the
5. Results Interpretations
61
protein presents a single cooperative unit. Later Privalov (Privalov 1982) showed that
collagen doesn’t present a single cooperative unit. This indicates that collagen melting is
an extremely cooperative process and the number of residues forming a “cooperative
block” that melts as a single structural unit can be found from the ratio between the van’t
Hoff enthalpy and the calorimetric enthalpy. Privalov proposed that the denaturation of
collagen is a slow process. Therefore calorimetric studies of collagen must be carried out
only at low heating rate, and the transition temperature can be obtained by extrapolating
to zero heating rate. Davis and Bächinger (Davis and Bachinger 1993) examined the
triple helix-coil transition of type III collagen solution using optical rotary dispersion and
ascertained that the transition is a reversible equilibrium process. They observed a
hysteresis in the helix-coil transition, where the midpoint of the refolding transition is 6-7
°C lower than that of the unfolding transition. They agreed that collagen, in their case the
cooperative length or block was 95 tripeptide units. Engel and Bächinger (Engel and
Bächinger 2000) examined Type III collagen solution and suggested that a strong
argument against the kinetic model of the collagen denaturation is the hysteresis
accompanied “reversibility” of the process, which they observed in their investigation.
They fitted their experimental data with the equilibrium model by least square fit with
equation 3, taking into consideration that 1=+ XX UN , neglecting the state F. They used
the equation of Gibbs free energy, equation (5.7) to evaluate the thermodynamic
quantities: 000 ln STHKRTG ∆−∆=−=∆ , (5.7)
where ∆S0 is the entropy of transition.
Leikina et al (Leikina, Mertts et al. 2002) suggested using combination of ultra-slow
differential scanning calorimetry (DSC) with isothermal circular dichroism for Type I
collagen solution that the collagen denaturation is reversible. They ascertained that
collagen type I is thermodynamically stable accompanied by large hysteresis with
equilibrium time from several hours (rats) to several days perhaps months or years
(humans) and the apparent irreversibility of collagen denaturation at the time scale of
several hours is simply manifestation of hysteresis. They proposed that denaturation is an
extreme slow equilibrium process and collagen is overheated by at least several degrees
above the equilibrium in the reported denaturation experiments. Denaturation of
5. Results Interpretations
62
overheated collagen occurs much faster than renaturation creating an appearance of an
irreversible rate limited process. Also Persikov et al (Persikov, Xu et al. 2004) have
observed the reversibility in the thermal transition of collagen-like peptides.
In contrast to the authors mentioned above, Miles (Miles 1993) and Miles et al (Miles,
Burjanadze et al. 1995) have shown that the thermal denaturation of collagen is governed
by irreversible rate process in which collagen is transformed to denatured state via a
highly temperature dependent kinetic rate constant, not by equilibrium thermodynamics,
as in equation (5.8).
NTkdtdN )(−= (5.8)
They examined the thermal stability of lens capsules (non-fibrillar Collagen IV) and rat
tail tendon (fibrillar collagen I) with differential scanning calorimetry (DSC). They
observed no evidence of denaturation endotherm after holding heat-denatured tendons for
up to five days as indication of the irreversibility in the short and medium term. Miles
observed that the native collagen content declined according to a first order kinetics after
storing lens capsules isothermally. He observed also that the rate constant was highly
temperature dependent increasing about one order of magnitude every 2.3 °C rise.
Recently Miles and Bailey (Miles and Bailey 2004) have examined solutions of collagen-
like peptides using DSC and shown also that the dentauration is kinetic controlled. In the
last study they showed that endotherms could be classified in one of three regions: the
equilibrium region, the mixed region or the rate (kinetic) region, dependent on the
polymer concentration and the scanning rate. They suggested that holding collagen at
constant temperature will not stop the denaturation as it would if the system were in
equilibrium. Also as an argument against the equilibrium model, they proposed that
reducing the temperature will continue the denaturation but with a slower rate, in contrast
to the equilibrium where some helix would be recovered. They concluded, in agreement
with other studies (Miles 1993), (Miles, Burjanadze et al. 1995), (Leikina, Mertts et al.
2002) that the denaturation temperature is increasing logarithmically with the scanning
rate. Therefore it is impossible to extrapolate to zero scanning rate to obtain the
hypothetical equilibrium temperature.
5. Results Interpretations
63
Miles and Bailey (Miles and Bailey 2004) ascertained that the validity of kinetic model
does not depend whether the triple helix is irrecoverable or recoverable, and the
renaturation of molecular type I collagen, if it is possible at all, is very slow in relation to
the time scale of experiment.
The aim of this work is to examine the effect of the conservation process on the thermal
stability of bovine pericardium and to analyze the results with the thermodynamic and
kinetic models found in the literature. To assess the thermal stability, measurements of
the fraction of denatured collagen (DC) after selective digestion with α-chymotrypsin
were performed.
For the investigation of kinetic and thermodynamic parameters, it was assumed that the
thermal denaturation consists of two steps one reversible step followed by an irreversible
one, as described in equation (5.1). It was assumed also that α-chymotrypsin digests both
U and F fractions leaving them together in the supernatant. Therefore a mathematical
equation was derived from equation 5.1, 5.3, 5.4 and 5.6 for the summation of U and F
and it was called S, where S the fraction in the supernatant, as following
dtdX
dtdX
dtdX FUS += (5.9)
dtXXd
dtdX NUF )1( −−
= . Taking into consideration that K
XX UN = yields:
UU X
KkK
dtdX
+−=
1 (5.10)
Substituting equations (5.6) and (5.10) in equation (5.9):
)1(1
)1(1 SUF
S XK
kKXXK
kKdt
dX−
+=−−
+= (5.11)
It is assumed that the chemical equilibrium of the reversible reaction UNK⇔ is a very
slow reaction in comparison to the irreversible reaction; consequently the rate of F
formation will be determined by apparent first order reaction.
FNK
→/
(5.12)
For this purpose, isothermal experiments of Tutoplast-processed and lyophilized
pericardium with time variation have been performed to determine /K .
5. Results Interpretations
64
Temperature (°C)
140 145 150 155 160 165 170 175 180
K´
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Tutoplast-processed
Native lyophilized
Figure 5.9: The temperature dependency of /K for lyophilized and Tutoplast-
processed bovine pericardium
It can be seen that the denaturation of the Tutoplst-processed pericardium is faster than
that for the lyophilized pericardium in the range of the examined temperatures (145-175
°C), as shown in fig 5.9. The /K values can give an indication about the progress of the
thermal denaturation and consequently the resistance to this denaturation. The /K value is
too low at 145 °C for both the lyophilized and the solvent because the reaction is too slow
at this temperature. Increasing the temperature to 165 °C leads to 4.5-fold increase of the /K value of the Tutoplast-processed, whereas the /K value of the lyophilized is
increased 1.3 fold. This indicates that the lyophilized pericardium is healthier and has
higher resistance to thermal denaturation than the Tutoplast-processed. The largest jump
in the /K value of the lyophilized pericardium is observed at 175 °C, the temperature
increase from 160 to 175 °C leads to 2.6-fold increase of the /K value; however it is still
far away from that of the Tutoplast-processed pericardium. The thermal denaturation of
the solvent-processed-pericardium is sharp due to the lower thermal stability and
5. Results Interpretations
65
associated with an activation energy 118.95 kJ/mol, whereas the thermal denaturation of
the lyophilized pericardium is associated with an activation energy 61.02 kJ/mol.
It was not possible to get useful information from the isothermal experiments performed
with the native pericardium because it contains high moisture content (≈ 85.7%) that
dominates the behavior of the thermal stability. It is found that the high water content
accelerates the thermal denaturation of the pericardium. The water content is normally
evaporated within the first 5-10 min, as shown in fig 5.10, within this time fast
denaturation takes place. As long as the water is evaporated, the denaturation proceeds
but with different kinetics.
Time (min)0 5 10 15 20 25
wei
ght l
oss
(%)
0
20
40
60
80
100
Figure 5.10: The time dependency of weight loss of native bovine pericardium at 105
°C
5.2.2 Measurements of isotonic shrinkage temperature
As discussed in the previous section, the moisture content is the deciding factor in
shaping the thermal stability during the measurements of DC. Therefore it was reasonable
to exclude the effect of the moisture content by performing measurements of isotonic
shrinkage temperature at fully hydrated conditions in water bath, in which only the
structure integrity and healthiness plays a role in shaping the thermal stability.
5. Results Interpretations
66
5.2.2.1 The results
4 (4×1.5 cm2) strips from the native, native lyophilized and Tutoplast-processed
pericardium were used for the measurements of shrinkage temperature. The Tutoplast-
processed pericardium starts to shrink approximately from 42 °ّC until the maximum
shrinkage is reached at 70 °C, as shown in fig 5.11. The lyophilized and the native
pericardium have almost similar progress in the shrinkage curve. The shrinkage intervals
are (65-73 °C) and (64-72 °C) respectively.
Temperature (°C)
20 30 40 50 60 70 80
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100
processed native lyophilized
native
Figure 5.11: The shrinkage curve for the native, native lyophilized and Tutoplast-
processed pericardium (average of 4 samples, each curve contains
approximately 1000 points, 95% confidence interval lies between ± 2-
17)
5.2.2.2 Discussion of the results
There is no absolute shrinkage temperature (Weir 1949), the shrinkage temperature must
be defined at certain constant heating rate and constant isotonic load. Furthermore some
authors defined the shrinkage temperature as the temperature at which the shrinkage
5. Results Interpretations
67
begins (Lennox 1949), while others as the temperature at which the tissue shrinks to 50%
of its maximum shrinkage (Rasmussen, Wakim et al. 1964; Danielsen 1990). The
shrinkage process of Tutoplast-processed strips was slow and takes place over relatively
wide temperature range (42-70 °C). Therefore, considering the shrinkage temperature as
the temperature at which the tissue shrinks to 50% will overestimate the shrinkage
temperature of Tutoplast process pericardium. The presence of residual ions in the
Tutoplast-processed pericardium resulted from the processing was confirmed by the
measurements of the thermal conductivity; 5 (8×6 mm2) native samples in 30 ml water
had thermal conductivity 12.6 µs/sec, whereas the processed 138 µs/sec. The presence of
the residual ions leads to swelling of the tissues during the shrinkage process and
consequently to thick samples, which shrink too slowly.
Lennox (Lennox 1949) has examined the effect of moisture content on the shrinkage
temperature. He concluded that increasing the soaking period increases the moisture
content and reduce the shrinkage temperature. Furthermore Weir (Weir 1949) observed
that dry specimens elongate before shrinkage occurs, but after soaking 1 h or longer in
water no preliminary elongation was observed. Therefore, in this work in order to
exclude any effect of moisture content or any preliminary elongation, the strips were
rehydrated in 0.9% NaCl solution for 24 h to have fully hydrated strips.
The bovine pericardium is known to be mechanically anisotropic, which affects the
mechanical properties of the pericardium (Crofts and Trowbridge 1989; Lee, Haberer et
al. 1989). The mechanical anisotropy was proved to be irrelevant for the initiation of
thermal denaturation (Lennox 1949). In this study, despite the well-known non-uniform
orientation of the pericardium, the randomly chosen strips have almost the same
shrinkage temperature.
Regarding the extent of the shrinkage, Lennox (Lennox 1949) observed two different
groups with different extent of shrinkage along the fatty layers. In this study, the extent of
shrinkage is different from one sample to another and no conclusion can be drawn about
the effect of temperature or processing on the extent of shrinkage. Therefore the
shrinkage for each sample is related to the maximum shrinkage, which gives almost
homogenous results.
5. Results Interpretations
68
The results of shrinkage temperature confirmed the conclusion drawn from the
measurements of DC that the dehydration or the moisture content has governed the
thermal stability. Under fully hydrated conditions, the thermal stability or the
thermoelasticity of the Tutoplast-processed pericardium to resist shrinkage was relatively
low in comparison with that for the native and the native lyophilized pericardium. It is
believed that structural modification or destruction induced by the processing causes this
reduction of thermal stability.
A conclusion has been drawn during the measurements of DC that the native lyophilized
pericardium samples are relatively intact undamaged. This is drawn from the smooth
denaturation in the temperature range (135-170 °C) during the DC measurements. This
conclusion has been also verified by the shrinkage temperature measurements. The
shrinkage temperature or the shrinkage curve was almost similar to that of the native
pericardium. Both of them were thermally unaffected until 64 and 65 °C respectively and
for both of them, the shrinkage proceeds quickly within 5 degrees.
5.2.2.3 Modelling of the thermal shrinkage of pericardium
The shrinkage curve consists of three different regimes (Chen, Wright et al. 1997; Chen,
Wright et al. 1998): an initial slow shrinkage regime, a rapid, large-shrinkage regime, and
finally a slow continuing-shrinkage regime.
Weir (Weir 1949) assumed that the shrinkage of collagen is described by a first order
kinetics as the following:
kLdtdL
−= (5.13)
Rearranging and integrating yields:
∞∞ +−−= LkteLLL )( 0 (5.14)
where
L is the length of the tissue at time (t), ∞L the completely shrunken length, 0L the initial
length and k the shrinkage rate constant.
Plotting ln( ∞− LL ) against t will yield a straight line of slope –k. Weir (Weir 1949)
disagreed with the use of such relation to obtain the rate constant (k) due to the negligible
5. Results Interpretations
69
linearity at the initial region of the shrinkage curve. Instead of that, he suggested to use
the time of half-shrinkage t1/2.
2/1/2ln tk = (5.15)
The model described in equation (5.13) is absolutely empirical and doesn’t explicitly
describe the progress of the denaturation of collagen molecules. In the current work, an
alternative model has been developed to describe the isothermal and the non-isothermal
isotonic shrinkage of collagen, in which the progress of the shrinkage is described
mathematically more in details and related to the number of denatured collagen
molecules.
The progressive denaturation of individual molecules
It is assumed that the collagen fiber consists of n collagen molecules; and that all the
molecules have the same length l with the total fiber length L .
At the beginning, where no shrinkage or denaturation occurs, there are only native
molecules and therefore:
NlnL ⋅= 00 (5.16)
where
0n is the initial number of collagen molecules and Nl the length of native molecules
If denaturation starts at t > 0, the hydrogen bonds will be broken, some molecules will
become denatured, as shown in Fig. 5.12, and consequently the fiber will begin to shrink
having the length )(tL at time t:
DDNN lnlntL +=)( (5.17)
where Nn is the number of native molecules at time t, Dn the number of denatured
molecules at time t and Dl the length of the denatured molecules. It can be assumed that
the total number of the molecules within a fiber is always constant, and therefore:
DN nnn +=0 (5.18)
5. Results Interpretations
70
Figure 5. 12: Schematic representation of the native and denatured collagen. Nl and
Dl represents the length of the native and denatured collagen molecule
respectively
Combining equations (5.16-5.18) yields:
)()( 0 DND llnLtL −−= (5.19)
The last equation is valid for both the isothermal and non-isothermal shrinkage of
collagen.
Modeling of isothermal shrinkage
As previously assumed (Weir 1949; Miles 1993; Miles, Burjanadze et al. 1995), the
shrinkage or denaturation of collagen follows first order kinetics, rearranging equation
(5.13) in terms of n:
nTkdtdn )(−= (5.20)
Integrating equation (5.20) yields:
tTkennN)(
0−= (5.21)
Taking equation (5.18) into consideration:
))(1(0tTkennD
−−= (5.22)
Substituting equation (5.22) in equation (5.19) gives:
Nl
Dl
5. Results Interpretations
71
))()(1()( 00 DN lltTkentLL −−−=− (5.23)
The last equation describes the time-dependence progress of shrinkage mathematically.
The t1/2 is defined at 50% of the maximum shrinkage. Therefore it is reasonable to relate
the shrinkage at time t to the maximum shrinkage at ( ∞t or endt ):
∞∞−−
−−=
−−
tTke
tTketLLtLL
)(1
)(1)(
)(
0
0 (5.24)
Due to the asymptotic behavior of the shrinkage curve, ∞t can be defined as the time at
which the shrinkage rate is reduced to 1% of its rate in the linear region, therefore
equation (5.23) is differentiated to obtain the rate of the shrinkage and consequently:
01.0)(
0
=−=⎟⎠⎞
⎜⎝⎛
⎟⎠⎞
⎜⎝⎛
∞∞ tTke
dtdLdtdL
t
t (5.25)
In this case it is assumed that 0t is the time at which the linear region of the shrinkage
curve begins. Rearranging equation (5.25) yields an equation for the determination of ∞t :
)(100lnTk
t =∞ (5.26)
The time of maximum shrinkage ∞t can related to the time of the half shrinkage 2/1t :
2/164.6 tt =∞ (5.27)
Temperatures 65, 66.5, 68, 69.5 °C are selected to perform the isothermal experiments
with the bovine pericardium. These temperatures are selected based on previous
examinations in the last section, which showed that these temperature lie in the shrinkage
range of the pericardium. The current results prove that the shrinkage of pericardium is
kinetically controlled, the higher the temperature, the faster the shrinkage. Assuming that
the shrinkage follows first order kinetics, the time of the half shrinkage (t1/2) is used for
the determination of the kinetic constant (k), as shown in table 5-1.
5. Results Interpretations
72
Table 5-1: The kinetic parameters of the mean shrinkage curve of the native
pericardium (4-5 samples)
Temperature (°C) t1/2 (sec) k (sec-1)
65 385 0.0018
66.5 64 0.0108
68 32 0.0221
69.5 25 0.0277
For the determination of the kinetic parameters, Arrhenius equation was used:
)/exp( RTEAk −= (5.28)
Plotting ln (k) against (1/T), as shown in Fig 5.13, yields the slope (E/R) and the intercept
(ln A). The activation energy, E, and the frequency factor, A, for the bovine pericardium
examined in this study are 565.95 kJ/mol and 8.64×1084 sec-1 respectively.
1/T*103 (1/K)
2.91 2.92 2.93 2.94 2.95 2.96 2.97
ln (k
)
-8
-7
-6
-5
-4
-3
-2
Figure 5.13: Plot of the natural logarithm of the kinetic constant k against the
inverse of the temperature (error bars indicate 95% confidence interval
for 4-5 samples)
5. Results Interpretations
73
The current values lie close to the kinetic parameters of collagen denaturation reported in
the literature, which supposed to be different from tissue to another. The E values in the
literature vary between 102-103 kJ/mol, whereas A between 1030-10105 sec-1 (Vijverberg,
Pearce et al. 1993; Agah, Pearce et al. 1994; Pearce and Thomsen 1995; Moran,
Anderson et al. 2000).
The experimental data obtained from the different isothermal curves are modeled using
equation (5.24). The proposed model has fitted the experimental data well, as shown in
Fig 5.14. The largest deviation is observed at lower temperatures. This could be attributed
to the relatively long lag phase or initial shrinkage region. As a limitation of the model,
the delay or the initial slow region at low temperatures can’t be modeled or fitted well
because the model describes the progress, as one single step, exponentially. It is
concluded that the model fits the higher temperatures better and the deviation becomes
almost negligible because the initial region is too short at higher temperatures to be
detected.
Time (sec)0 200 400 600 800 1000 1200 1400
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100
65 °C
66.5 °C
68 °C
ModelExperimental
Figure 5.14: The measured and modeled isothermal shrinkage curve of the bovine
pericardium (average for 5 samples)
5. Results Interpretations
74
Modeling of non-isothermal shrinkage
In the normal shrinkage temperature experiment, the temperature changes with time
according to a constant heating rate (q). Therefore rearranging equation (5.13):
nTkqdT
dn )(1−= (5.29)
)/exp()( RTEATk −= (5.30)
∫ −−=∫T
T
n
ndTRTE
qA
ndnN
00
)/(exp (5.31)
where, A is the frequency factor and E the activation energy
The integration of the term dTRTE∫ − )/(exp in equation (5.31) can be expressed in the
form (Senum and Yang 1977):
[ ])()()/(exp 0xfxfREdTRTE −=∫ − (5.32)
where RTEx /=
There are different degrees of rational approximations for )(xf (Senum and Yang 1977).
The first degree of approximation is considered as the following:
21.)exp()(+
−=
xxxxf (5.33)
Assuming that )( 0xf is negligible, therefore:
⎥⎦⎤
⎢⎣⎡
+−−
=2/
1./
)/(expln0 RTERTE
RTERE
qA
nnN (5.34)
)2/
)/(exp(exp0 ⎥⎦⎤
⎢⎣⎡
+−−
=RTE
RTEqTAnnN (5.35)
))2/
)/(exp(exp1(0 ⎥⎦⎤
⎢⎣⎡
+−−
−=RTE
RTEqTAnnD (5.36)
Substitution of equation (5.35) in equation (5.19) yields:
)())2/
)/(exp(exp1()( 00 DN llRTE
RTEqTAnTLL −⎥⎦
⎤⎢⎣⎡
+−−
−=− (5.37)
The last equation describes the temperature-dependence progress of the shrinkage of
collagen. Analogously to the isothermal shrinkage, the shrinkage at temperature T is
related to the maximum shrinkage at temperature ∞T :
5. Results Interpretations
75
)2/
)/(exp(exp1
)2/
)/(exp(exp1
)()(
0
0
⎥⎦
⎤⎢⎣
⎡+
−−−
⎥⎦⎤
⎢⎣⎡
+−−
−=
−−
∞
∞∞∞
RTERTE
qAT
RTERTE
qTA
TLLTLL (5.38)
Some authors defined the shrinkage temperature as the temperature at which the
shrinkage begins (Lennox 1949), while others as the temperature at which the tissue
shrinks to 50% of its maximum shrinkage (Rasmussen, Wakim et al. 1964; Danielsen
1990).
The experimental data obtained from the non-isothermal shrinkage curve are fitted with
the proposed model using equation (5.38), after the determination of E and A from the
isothermal experiments. The suggested model has also fitted the experimental data
satisfactorily, as shown in Fig 5.15. Small deviation from the experimental data is also
observed at the initial and final regions. The shrinkage temperature, taken at 50% of the
maximum shrinkage, is 68.0 °C.
Temperature (°C)40 50 60 70 80
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100
Model
Experimental
Figure 5.15: The measured and modeled non-isothermal shrinkage curve of the
bovine pericardium under heating rate 2.5 °C/min (average for 5
samples)
5. Results Interpretations
76
Little deviation is observed at the initial region as well as at the final slow continuing
shrinkage region. The model, based on the kinetic parameters obtained from the
isothermal experiments, fits a one-step shrinkage curve with an exponential form, which
deviates slightly from the measured shrinkage curve, due to the relatively long initial and
final regions observed with the measured curve.
The denaturation or shrinkage temperature of collagen is heating rate dependent (Weir
1949; Miles 1993; Miles, Burjanadze et al. 1995), therefore it is reasonable to establish a
relationship between the heating rate and the shrinkage temperature. For this purpose, a
dimensionless heating rate parameter is introduced, EAqRB = in addition to the
dimensionless temperature parameterRTEx = . Rearranging equation (5.38) yields
)2
)(exp1(exp1
)2
)(exp1(exp1
)()(
0
0
⎥⎦
⎤⎢⎣
⎡+−−
−
⎥⎦⎤
⎢⎣⎡
+−−
−=
−−
∞
∞
∞
∞
xx
Bx
xx
xBTLLTLL (5.39)
Introducing ⎥⎦⎤
⎢⎣⎡
+−
=2
)exp(1x
xx
z results in:
)/exp(1)/exp(1
)()(
0
0
BzBz
TLLTLL
∞∞ −−−−
=−− (5.40)
The shrinkage temperature T50 is defined as the temperature at which the tissue shrinks to
50% of its maximum shrinkage. Therefore, substituting 0.5 in the left side of equation
(5.40) under constant B leads to the determination of 2/1z and consequently 2/1x .
Variation of the heating rate, q , and consequently B leads to variation of 2/1z and 2/1x .
A relationship between 2/1x and B , which shows the effect of varying the heating rate in
the range (0.1-50 °C/min).on the shrinkage temperature, is shown in Fig 5.16.
5. Results Interpretations
77
B=qR/EA *1090 (-)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
x 1/2
=E/R
T 50
(-)
196
197
198
199
200
201
202
203
204
0.1
2
5
502520
10
Figure 5.16: The influence of varying the heating rate parameter B on the
temperature parameter x in the range of heating rate (0.1-50 °C/min)
An exponential-like relationship has been observed. It is seen that the shrinkage
temperature is sensitive to the change of heating rate especially at lower heating rates.
The largest drop in temperature parameter 2/1x is observed in the range (0.1-2 °C/min),
i.e. the shrinkage temperature is increased approximately by five degrees, from 62.5 to
67.4 °C. Afterwards, with increasing the heating rate the shrinkage temperature will
increase but not as significant as the case at lower heating rates. Interestingly, the
modelled shrinkage temperature at the heating rate used in the current work, 2.5 °C/min,
taken from fig 5.16 is very close to the experimental shrinkage temperature. The
modelled shrinkage temperature is 67.8 °C, whereas the experimental is 68 °C.
5. Results Interpretations
78
5.2.3 SDS-PAGE investigations
As discussed previously, thermal denaturation induces unfolding of triple helix into
random coils, which makes collagen susceptible for the enzymatic degradation. SDS-
PAGE was used to evaluate the thermal stability of collagen by analyzing the α-
chymotrypsin digested fraction of collagen after the thermal treatment. Determining the
fragments size during the SDS-PAGE investigation can provide information about the
extent of denaturation.
5.2.3.1 Results
Figure 5.17 shows SDS-PAGE of native and Tutoplast-processed pericardium at room
temperature and 55 °C, taken from (Koerber 2006). At room temperature, clear large
bands or fragments are observed in the range of 100, 66, 55, 13, 15 and 10 kD with the
native pericardium, whereas only band in the range of 150 kD is detected with the
Tutoplast processed pericardium.
At 55 °C the native pericardium has weak bands in the range of 120, 97, 22 and 14 kD,
whereas the Tutoplast processed pericardium has the same fragment, 150 kD, found at
room temperature.
Figure 5.17: SDS-PAGE for standard (lane 1), native pericardium at room
temperature (2, 3), processed at room temperature (4, 5), native at 55
°C (6, 7), processed at 55 °C (8, 9) and α-chymotrypsin (10)
5. Results Interpretations
79
The number of large fragments becomes less for the native pericardium at 90 °C, as
shown in fig 5.18. Bands in the range of 31, 21.5, 15 and 13 kD were detected. For the
Tutoplast-processed pericardium, as observed at room temperature and 55 °C, only a
large fragment at 110 kD is observed.
The bands, observed with the native pericardium at 90 °C, are also detected at 120 °C but
with lower intensity. No further large fragments have been detected at 120 °C for the
Tutoplast-processed pericardium.
Figure 5.18: SDS-PAGE for standard (lane 1), native pericardium at 90 °C (2, 3),
processed at 90 °C (4, 5), native at 120 °C (6, 7), processed at 120°C (8,
9) and α-chymotrypsin (10)
No large fragments were detectable neither with the native nor with the Tutoplast-
processed pericardium at temperatures 135 and 150 °C, as shown in fig 5.19.
5. Results Interpretations
80
Figure 5.19: SDS-PAGE for standard (lane 1), native pericardium at 135 °C (2, 3),
processed at 135 °C (4, 5), native at 150 °C (6, 7), processed at 150°C (8,
9) and α-chymotrypsin (10)
Also no large fragments were detectable neither with the native nor with the Tutoplast-
processed pericardium at temperatures 160 and 170 °C, as shown in fig 5.20.
5. Results Interpretations
81
Figure 5.20: SDS-PAGE for standard (lane 1), native pericardium at 160 °C (2),
processed at 160 °C (3), native at 170 °C (4, 5), processed at 170°C (6,
7), native at 185 °C (8, 9) and α-chymotrypsin (10)
5.2.3.2 Discussion
The denaturation of collagen triple helix occurs through a melting of the hydrated
crystallites, involving rupture of hydrogen bonds and rearrangement of the triple helix
into random chain configuration (Miles and Bailey 2001). The denaturation of collagen
increases the susceptibility to proteolytic degradation. During the denaturation the triple
helix unwinds leaving the cleavage sites accessible for enzymatic degradation. SDS-
PAGE separates the proteins or the fragments according to their size. The determination
of the fragment size for enzymatic digested proteins gives an impression how accessible
the cleavage sites were.
Regarding the SDS-PAGE at room temperature, large fragments have been detected with
the α-chymotrypsin digested fraction of native pericardium. It is expected that these
fragments represent the soluble and the new synthesized non cross-linked collagen, which
α-chymotrypsin can digest (Bank, Krikken et al. 1997). In the case of the Tutoplast-
5. Results Interpretations
82
processed pericardium, it is assumed that the soluble proteins are already eliminated
during Tutoplast-process (Schoepf 2006). This explains the absence of fragments on the
gel except only one band in the range of 150 kD. This band could represent the processed
and modified terminal domains outside the triple helix.
At 55 °C the number of large fragments in the native pericardium becomes less.
According to the measurements of DC discussed in section 5.2.1.2, the denaturation of
native pericardium starts at 55 °C. It is supposed that at this temperature, domains of the
triple helix start to unfold through heating, which makes them susceptible to α-
chymotrypsin. It is supposed that no large fragments of the soluble proteins still
detectable. In the Tutoplast-processed pericardium, the band is the same as that obtained
at room temperature. A simple explanation for this behavior is that the terminal domains
still resistant at this temperature against heating but can be digested in large fragments by
α-chymotrypsin. The triple helix, according to the results of DC measurements and the
effect of dehydration discussed previously, can’t be attacked yet during the heating and
consequently it is not susceptible to α-chymotrypsin.
At 90 °C the denaturation goes on with the native pericardium allowing more cleavage
sites to be susceptible to α-chymotrypsin leading to the reduction of large fragments in
the SDS-PAGE. For the Tutoplast processed pericardium, the terminal collagen starts to
be affected from heating causing fragmentation and consequently leading to smaller
fragments, however still large. Also the triple helix is still not attacked.
At 120 °C, in consistency with the measurements of DC, the denaturation reaches almost
close to the maximum denaturation with the native pericardium. More cleavage sites
become accessible for α-chymotrypsin, which lowers the intensity of the large fragments.
For the Tutoplast-processed no large fragments are detectable. It is expected that the
terminal domains are extremely denatured and fully digested by α-chymotrypsin. It is
thought also that the triple helix in not affected.
No fragments are detected at temperatures 135 and 150 °C, neither for the native nor for
the Tutoplast-processed. It is assumed at these temperatures that the triple helix of the
native pericardium is fully accessible for α-chymotrypsin, and the triple helix of the
Tutoplast-processed pericardium is still not accessible, which explains the absence of
large fragments on the SDS PAGE.
5. Results Interpretations
83
No large bands are detected in the range 160-185 °C, neither with the Tutoplast-
processed nor with the native pericardium.
The absence of bands is observed during the SDS-PAGE investigations of the Tutoplast
processed pericardium either at low temperatures or at high temperatures (above 160 °C).
It is not easy, using SDS-PAGE only, to judge how the extent of denaturation is, because
during the investigations of SDS-PAGE, only the denatured fractions are tested without
obtaining any information about the intact fractions. Consequently, the analysis and the
assessment of the SDS-PAGE investigation could be better understandable with the help
of the previous analysis of the measurements of DC.
During the measurements of DC for the Tutoplast-processed pericardium, The DC is
almost unchanged until 150 °C, thereafter, sharp transition from the triple helix to
random coils is observed in the range 160-170 °C. Therefore, the absence of large
molecules in the Tutoplast-pericardium could be attributed to the fully inaccessible
cleavage sites at temperatures up to 160 °C, and to the fully accessible cleavage sites at
temperatures above 160 °C.
5.3 The effect of different steps in the Tutoplast® process The analysis of the results of the thermal stability draws the attention to many crucial
conclusions, which can help to understand the effect induced by Tutoplast-process. It was
concluded the processing induces two contradictory factors, stabilizing factor,
represented by the dehydration, and destabilizing factor, which may represented by
structural modification. The next challenge was to investigate the role or the effect of
each step of Tutoplat process and its contribution in the structural modification caused by
the process.
5.3.1 The effect of sodium hydroxide treatment
The NaOH treatment in the process is scientifically recognized as an acceptable and
effective methodology for reducing prion infectivity by six log (Brown, Rohwer et al.
1986; Schoepf 2006). However treating the collagenous tissues with NaOH leaves
detrimental side effects (Kearney and Johnson 1991).
5. Results Interpretations
84
5.3.1.1 The hydrolysis of collagen amino acids
The extent of hydrolysis at room temperature was checked by measuring the amount of
the aromatic amino acids dissolved in the NaOH solution spectrophotometrically at 280
nm.
First a calibration curve for the content of aromatic amino acids is constructed using a
stock solution of 2 mg aromatic amino acids in 100 ml water, as discussed in section
4.3.2.1. Different dilution series have been performed to obtain the calibration line shown
in fig 5.21 (Koerber 2006).
m AA (µg)
0 5 10 15 20 25
Abs
orba
nce
280
nm (-
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Figure 5.21: The calibration line of the aromatic amino acids. The absorbance
measured at 280 nm, error bars indicate 95% confidence interval for 5
samples (y = 0.0259 x + 5.46.10-3, r2 = 0.9964)
The amount of collagen dissolved or hydrolyzed in the NaOH solution can be
determined, taking into consideration that the aromatic amino acids constitute 2.84 % of
the collagen (Fietzek and Kuehn 1976).
Fig 5.22 shows the time-dependent hydrolysis of the collagen in the native pericardium at
room temperature. The amount of dissolved or hydrolyzed collagen is increased linearly
5. Results Interpretations
85
with the time of treatment. However, the amount is too low. Treating the native
pericardium with 1 N NaOH for 150 min yields an average amount of dissolved collagen
of 137 µg, which is lower than 1% of the original dry weight of the sample.
Time (min)
0 20 40 60 80 100 120 140 160
mc
diss
olve
d (%
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Figure 5.22: Time-dependent hydrolysis of collagen during the treatment of native
pericardium with 1 N NaOH at room temperature, error bars indicate
95 % confidence interval for 10 samples
The same linear time-dependence hydrolysis of collagen is almost observed with the
Tutoplast-processed pericardium samples, as shown in fig 5.23. Also the amount of
dissolved collagen in the NaOH solution is neglected in comparison with the original dry
weight.
5. Results Interpretations
86
Time (min)
0 20 40 60 80 100 120 140 160
mc
diss
olve
d (%
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Figure 5.23: Time-dependent hydrolysis of collagen during the treatment of
processed pericardium with 1 N NaOH at room temperature, error bars
indicate 95 % confidence interval for 10 samples
5.3.1.2 The measurements of the shrinkage temperature
For the assessment of the effect of the NaOH solution treatment and the subsequent
neutralization step on the quality of native bovine pericardium, measurements of isotonic
shrinkage temperature were used and analyzed.
5.3.1.2.1 The effect of NaOH solution
The destruction or the structural modification induced by the NaOH treatment is
obviously illustrated by the extreme reduction of the shrinkage temperatures or the
thermoelasticity to resist the shrinkage, as shown in fig 5.24. The NaOH treated samples
starts to shrink approximately at 42 °C, whereas the control samples at 67.5 °C. The
shrinkage of the swelled NaOH-treated samples is slow and takes place over a relatively
wide temperature range (~ 30 degrees). The shrinkage process of the native untreated
samples is too fast and occurred mostly within 5 degrees.
5. Results Interpretations
87
20 30 40 50 60 70 80 90
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100
NaOH
native
Figure 5.24: The influence of 1 N NaOH treatment on the shrinkage temperature of
the pericardium (average of 5 samples)
5.3.1.2.2 The effect of the neutralization step
The efficiency of acetic acid treatment to restore the tissues to their physiological state
was tested. Fig 5.25 shows the influence of treating the pericardium strips with 1 N
CH3COOH after the 1 N NaOH treatment. It is observed that the 5-min treatment results
in tissues with pH of 5 and thermal shrinkage starting from 50 °C, whereas the 15 min-
treatment yields tissues with pH 3 and thermal shrinkage starting from 45 °C.
5. Results Interpretations
88
Temperature (°C)
20 30 40 50 60 70 80 90
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100
5 min
15 min
Figure 5. 25: The effect of varying the duration of the acetic acid neutralization step
on the shrinkage temperature of NaOH-treated pericardium (average
for 5 samples)
It was reasonable to check the effect of lowering the CH3COOH concentration. Under
constant treatment duration, 15 min, the 0.1 N CH3COOH leaves tissues with a pH 6 and
thermal shrinkage starting from 62 °C, where as the 1 N CH3COOH results in tissues
with pH 3 and thermal shrinkage starting from 45 °C, as shown in fig 5.26.
5. Results Interpretations
89
Temperature (°C)
40 50 60 70 80 90
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
1001 N CH3COOH
0.1 N CH3COOH
Figure 5.26: The effect of varying the concentration of the acetic acid neutralization
step on the shrinkage temperature of NaOH-treated pericardium
(average of 5 samples)
As alternatives to acetic acid, the influence of rinsing solutions to restore the tissues to
the physiological state was tested. Immersing the NaOH-treated strips in distilled water
(pH = 7.8) without stirring for 30 min does nothing to reduce the pH value of the strips.
The strips preserve their pH value 14 with thermal shrinkage starts at 48 °C, as shown in
fig 5.30. The capacity of phosphate buffer (pH = 7.4) is insufficient to reduce the pH
value of the strips. After 30 min stirring in 100 mmol phosphate buffer (pH 7.4), the final
pH of strips is 10.5. The thermal shrinkage starts at 54 °C. Fig. 5.27 shows that stirring
the tissues in distilled water (pH = 7.8) for 90 min, in which water is changed every 15
min, is the best variant to treat the tissues. The strips have a final pH value of 8.0 and
thermal shrinkage starting at 62 °C.
5. Results Interpretations
90
Temperature (°C)
20 30 40 50 60 70 80 90
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
100stirred/changed water 75 min
stirred phosphate buffer 30 min
immersed in water 30 min
Figure 5.27: The effect of different buffering systems on the shrinkage temperature
of NaOH-treated pericardium (average of 5 samples)
Figure 5.28 shows that treating the NaOH-treated strips with 1 N CH3COOH followed by
1-2 10-min water or phosphate buffer baths results in tissues with shrinkage curve close
to that one of the native untreated samples. The tissues begin to shrink from 63 and 64 °C
after the CH3COOH/phosphate buffer and CH3COOH/distilled water treatment
respectively, whereas 65 °C for the native untreated samples, as discussed in section
5.2.2.
5. Results Interpretations
91
Temperature (°C)
20 30 40 50 60 70 80 90
Nor
mai
lized
Shr
inka
ge (%
)
0
20
40
60
80
100
water
phosphate buffer
Figure 5.28: The Influence of the 0.1 N acetic acid treatment step followed by
distilled water or phosphate buffer bath (pH 7.4) on the thermal
shrinkage of NaOH-treated pericardium (average of 5 samples)
5.3.1.3 The measurements of DC
The measurements of DC, seen in fig 5.29, show no significant influence of the 1 N
NaOH treatment for 1 h. The native samples have DC, average ± 95 % confidence
intervals, 11.6 % ± 1.3, whereas the NaOH treated samples 12.0 % ± 3.3.
5. Results Interpretations
92
Native NaOH
DC
(%)
8
10
12
14
16
18
Figure 5.29: The influence of 1 N NaOH treatment for 1 h on the DC of bovine
pericardium, error bars indicate 95 % confidence interval for 5 samples
5.3.1.4 SDS-PAGE investigations
The α-chymotrypsin digested fraction of NaOH-treated pericardium was taken for the
SDS-PAGE investigation. Regardless of the concentration or the duration of the NaOH
treatment, no large fragments can be detected, as shown in fig. 5.30 and 5.31, as
indication of the extreme denaturation
5. Results Interpretations
93
Figure 5.30: SDS-PAGE for standard (lane 1), 30 min 0.5 N NaOH-treated
pericardium (2, 3), 1 h 0.5 N NaOH-treated pericardium (4, 5), 2 h 0.5 N
NaOH-treated pericardium (6, 7), 30 min 1 N NaOH-treated
pericardium (8, 9) and α-chymotrypsin (10)
Figure 5. 31: SDS-PAGE for standard (lane 1), 1 h 1 N NaOH-treated pericardium
(2, 3), 2 h 1 N NaOH-treated pericardium (4, 5), 30 min 2 N NaOH-
treated pericardium (6, 7), 1 h 2 N NaOH-treated pericardium (8, 9)
and α-chymotrypsin (10)
5. Results Interpretations
94
5.3.1.5 Discussions
The treatment of pericardium strips with NaOH results in swelling of the strips, as shown
in fig 5.32.
Figure 5.32: Swelled NaOH-treated bovine pericardium
The swelling of collagen and other fibrous proteins in acid and alkaline solutions is
governed by the osmotic pressure differences arising between the protein phase and the
external solution as a result of the formation of protein salts (Donnan membrane effect)
and by the cohesion of the protein i.e. the forces opposing swelling, such as interweaving
of the fibres and intermolecular forces, first observed by (Procter 1914). The degree of
swelling will depend on the balance of these two factors. In contrast to the acid swelling,
which decreases below pH 2.0, the alkaline swelling increases progressively with
increasing pH and shows no decrease at high pH (Bowes and Kenten 1950). The increase
in alkaline swelling at high pH could be attributed to the reduction of cohesion forces as a
result of breaking of structural features (Bowes and Kenten 1950).
The alkaline hydrolysis of collagen at high temperatures is a well-known method for a
complete degradation or hydrolysis (Kang, Dixit et al. 1975; Mann, Gaill et al. 1992).
However very little attention has been paid to the partial degradation of proteins by low
concentrations of alkali at low temperatures (Berry, Hong et al. 1989). In the current
work the extent of collagen hydrolysis in 1 N NaOH treated native as well as Tutoplast-
Swelled Native
5. Results Interpretations
95
processed pericardium samples at room temperature was checked based on the content of
the aromatic amino acids. The analysis of the results shows a time-dependent hydrolysis
of the collagen in the NaOH solution; however the content of dissolved or hydrolyzed
collagen is low. After 150 min treatment the amount of dissolved collagen is lower than 1
% of the original dry weight. These results confirm with previous investigations that
showed that NaOH could hydrolyze the intra- and intermolecular cross-links without
destroying the helical structure of the collagen molecule (Fujii 1969; Hattori, Adachi et
al. 1999). Kearney and Johnson (Kearney and Johnson 1991) extracted 55.6 µg/mg dry
weight collagen during 18 h treatment with 1 N NaOH, which is considered as low
content of extracted collagen.
The shrinkage temperature analysis shows an extreme reduction in the thermal stability or
in the thermoelasticity to resist the shrinkage. The NaOH-treated strips start to shrink at
42 °C, whereas the native untreated strips at 67.5 °C. Furthermore the shrinkage of the
NaOH-treated strips is too slow and occurrs over wide temperature range, approximately
30 degrees, because the strips are thick and swelled from the action of the alkali, which
prevents smooth shrinkage of the strips. This reduction of the thermal shrinkage is
expected due the well-known action of alkali on the collagen, represented by amino acids
modification and destruction of intra-and intermolecular collagen cross-links. This alkali
action is explained in details in the literature; Hattori et al (Hattori, Adachi et al. 1999)
have shown that the extractability of collagen by the alkaline treatment for 14 day at
room temperature was much higher than that by pepsin or acetic acid, especially from an
aged samples. They justified the higher extractability by the ability of alkaline treatment
to remove the telopeptide involved in cross-linking of collagen molecule and to break
some additional cross-links in the triple helical region, which resistant to the pepsin
treatment. Previous studies (Rauterberg and Kuhn 1968; Fujii 1969; Hattori, Adachi et al.
1999) proved amino acid modifications induced by the alkaline treatment represented by
the deamination of acid amides of Asn and Gln. Hattori et al (Hattori, Adachi et al. 1999)
detected that the isoelectric point of collagen was lowered from 9.3 to 4.8 because of the
conversions of Asn and Gln to Asp and Glu, which reduced the thermal stability of
collagen.
5. Results Interpretations
96
The damage caused by NaOH treatment could be reversed or eliminated by a following
and immediate treating step.
After treating 10 pericardium strips in 100 ml 1 N NaOH, the NaOH solution is decanted
and the strips were treated with 1 N CH3COOH, 5 strips of them with 50 ml for 5 min
and the other 5 strips with 50 ml for 15 min. The aim of this treatment was to restore the
pericardium strips to their physiological state and pH. It is seen that this treatment shifts
the pH value of the strips too rapidly from the basic to the acidic region causing damage
also. 5 and 15 min treatment with 50 ml 1 N CH3COOH is enough to shift the pH to 5
and 3 respectively yielding pericardium strips with thermal shrinkage starting at 50 and
45 °C respectively. Not only alkaline reduces the thermal stability of collagen but also
acids because it cleaves hydrogen bonds (Gustavison 1956). The volumes of NaOH as
well as of CH3COOH used to treat the strips are chosen to have fully submerged strips.
For the treatment of 10 and 5 strips, 100 and 50 ml are used respectively. Therefore lower
concentrations of CH3COOH not lower volumes are further tested to treat the strips
without inducing secondary damage. 0.1 N CH3COOH has been used to treat the NaOH-
treated tissues. The 15-min treatment with 0.1 N CH3COOH shifts the pH value of the
pericardium strips to 6, resulting in thermal shrinkage starts at 62 °C, which is close to
that of the native untreated pericardium.
Also as alternative to the acid treatment, the efficiency of different washing or rinsing
fluids, such as distilled water and phosphate buffer (pH 7.4) has been tested. It is
observed that submerging or washing the NaOH-treated samples once with water or
phosphate buffer is not sufficient to reduce the pH value of the samples due to the limited
capacity of the water or buffer to wash the ions. A complete neutralization of the samples
could be achieved by intensive washing with distilled water baths for 90 min, in which
the water bath has to be changed every 10 or 15 min.
The best variant has been achieved by treating the NaOH treated strips with 1 N
CH3COOH for 15 min followed by one or two 10-min distilled water washing bath. With
this variant, the pericardium strips has a final pH value 8, the same pH as distilled water,
and a thermal shrinkage starts at 64 °C.
The idea of treating the damage induced by the alkaline treatment is well known in the
literature and has been borrowed from the treatment of skin or eye alkaline burns. The
5. Results Interpretations
97
damage caused by alkaline agent continues until the pH values returns to a physiological
level (Gruber, Laub et al. 1975). The use of acid neutralization in the treatment of skin
alkaline treatment is debatable. Some authors (Bromberg, Song et al. 1965; Wolfort,
DeMeester et al. 1970; Mozingo, Smith et al. 1988) suggested that alkaline burns should
be treated by water irrigation alone and the using of acid neutralization step to treat skin
alkaline burns produces exothermic reaction with the alkali that may extent the depth of
skin burn by increasing the temperature. Recently Andrews et al (Andrews, Mowlavi et
al. 2003) challenged the belief that the acid neutralization step causes secondary tissue
damage. They demonstrated using Wistar rats that the neutralization with acetic acid
offers a more rapid return to physiologic pH, less severe tissue damage, and improved
wound healing in comparison to those treated with water. Furthermore, they recorded the
skin temperatures that were below the rat body temperature, 33°C, from induction to
completion of the experiment. They suggested that although an exothermic reaction may
have been present, any heat produced was dissipated by the hypothermic room
temperature, 25°C, of the solutions used.
In the current work, it is observed that only washing with water to restore the tissues to
their physiological state takes long time and therefore an acetic acid treatment would be
helpful for quick restoration of the tissues but with moderate concentration and duration
to avoid a secondary damage.
The measurements of DC show no significant influence of the NaOH treatment. This
could be attributed to the fact that α-chymotrypsin is not active to digest collagen at
highly alkaline conditions and only active at neutral to slightly alkaline ranges (Moe and
Birkedal-Hansen 1979). Washing the samples with water until neutralization before being
digested with α-chymotrypsin is not a practical solution because as observed with the
shrinkage temperature measurements, the effect of NaOH is reversible by restoring the
tissues to their physiological neutral state.
Regarding the SDS-PAGE investigation, no large fragments are detectable with the α-
chymotrypsin digested fraction of the NaOH-treated samples. This gives indication how
strong the denaturation of collagen is and how accessible almost the cleavage sites for α-
chymotrypsin are. These results are consistent with previous study (Berry, Hong et al.
5. Results Interpretations
98
1989), which shows that treating collagen starting with a concentration of 0.25 N NaOH
yields heterogeneous small peptides (<20 kD).
5.3.2 The effect of hydrogen peroxide treatment
This step in the Tutoplsat® process has been confirmed to activate viruses, including
enveloped and non-enveloped DNA and RNA viruses (Schoepf 2006). However, the
oxidation with H2O2 leads to the modification of protein amino acids (Neumann and
Timasheff 1972).
5.3.2.1 The shrinkage temperature measurements
It is seen that the 3% treatment caused a little reduction in the thermal shrinkage, as
shown in fig 5.33. The 3%-treated pericardium starts to shrink from 60 °C, whereas the
native untreated samples at 66 °C. Furthermore, the shrinkage curve of the 10% treated
pericardium is almost very close; it starts to shrink at 58 °C.
Temperature (°C)
40 50 60 70 80 90
Nor
mal
ized
Shr
inka
ge (%
)
0
20
40
60
80
10010%
3%
w/o
Figure 5.33: The Influence of the varying the H2O2 concentration on the shrinkage
curve of the pericardium (average of 4 samples)
5.3.2.2 Measurements of DC
DC measurements show no significant influence of the H2O2 treatment regardless of the
concentration, 3, 10 and 30%, as shown in fig 5.34. The native untreated samples have
5. Results Interpretations
99
DC, average ± 95 % confidence interval, 11.6 ± 1.3, whereas the 3, 10 and 30% treated
samples 11.4 ± 3.3, 10.5 ± 3.8 and 9.9 ± 3.7 respectively.
Native 3% 10% 30%
DC
(%)
0
2
4
6
8
10
12
14
16
Figure 5.34: The influence of H2O2 treatment of the DC of native bovine
pericardium, error bars indicate 95 % confidence intervals of 5 samples
5.3.2.3 SDS-PAGE investigations
Native as well as Tutoplast-processed samples were treated with 3, 10 or 30% H2O2 and
digested overnight with α-chymotrypsin at 37 °C. The digested fraction was analyzed
using SDS-PAGE (Koerber 2006), as shown in fig 5.35.
Regarding the native 3%-treated sample, clear bands are detected in the range 97.4- 116.3
kD. Also weak bands are detected in the range of 40 and 56 kD, whereas with the
processed treated with 3%, only fragments larger than 100 kD are detected.
The native pericardium treated with 10% H2O2 is almost similar to the 3% treated
sample. It has large clear bands in the range of 100 kD and also weak bands in the range
between 45 and 56 kD. Large bands above 100 kD are observed with the processed 10%-
treated pericardium.
5. Results Interpretations
100
Regarding the 30% treatment, only clear bands in the range of 100 kD and weak bands
between 55 and 66 kD are found with the native pericardium.
Figure 5. 35: SDS-PAGE for standard (lane 1), 3% H2O2-treated native pericardium
(2), 3% H2O2-treated processed pericardium (3), 10% H2O2-treated
native pericardium (4), 10% H2O2-treated processed pericardium (5),
30% H2O2-treated native pericardium (6), 30% H2O2-treated processed
pericardium (7) and α-chymotrypsin (9)
5.3.2.4 Discussion of the results
The hydrogen peroxide treatment in the Tutoplast process has been found to be effective
against HIV (Hinton, Jinnah et al. 1992). However, the oxidation with H2O2 leads to the
modification of protein amino acids (Neumann and Timasheff 1972).
One of the most common pathways of protein degradation is the oxidation of amino acids
(Met, Tyr, Trp, Cys, and His). This oxidation can occur through photolytic or chemical
reactions, and is dependent on such factors as the temperature, pH, the presence of certain
excipients, heavy metals and the presence of molecular oxygen (Manning, Patel et al.
1989; Duenas, Keck et al. 2001). Hydrogen peroxide is a relatively nonspecific oxidizing
agent, which reacts with a wide variety of organic compounds. It can modify thioether,
5. Results Interpretations
101
indole, sulfhydryl, disulfide, imidazole and phenolic at the neutral or slightly alkaline
conditions (Manning, Patel et al. 1989). Under acidic conditions the primary reaction is
the conversion of methionine residues to sulfoxide (Neumann and Timasheff 1972).
Oxidation of Met residues is associated with the loss of the biological activity for many
proteins (Manning, Patel et al. 1989). Restoration of the biological activity was found to
be achieved with the reduction of Met sulfoxide to Met (Caldwell, Luk et al. 1978). In the
current work it is seen that the treatment with 3% H2O2 (pH = 5.52) is almost not
destructive to the collagen structure. This was confirmed by the high thermal stability
assessed by the measurements of isotonic shrinkage temperature. The samples treated
with 10% H2O2 (pH = 4.13) have almost similar thermal stability to those treated with
3% H2O2. In contrast to the 3 and 10% treatment, the treatment with 30% (pH = 2.40) is
extreme destructive and leads completely destructed pericardium strips, which can’t be
further tested with the isotonic shrinkage technique. It is expected the treatment with
H2O2 under mild concentrations and pH doesn’t lead to complete oxidation of the Met
residues and consequently to the complete destruction of the collagen, which is observed
at extreme concentration and pH.
Regarding the DC measurements, as discussed with the NaOH treatment, α-chymotrypsin
couldn’t attack the tissues under acidic conditions because it is inactive in this pH range.
SDS-PAGE Investigations are consistent with the measurements of the shrinkage
temperature. At mild concentrations, 3 and 10%, large fragments are detected with the α-
chymotrypsin digested fraction native pericardium as indication of the inaccessibility of
the cleavage sites to α-chymotrypsin. It is expected that the absence of large fragments
with the Tutoplast-processed pericardium could ascribed to the processing not to the
H2O2 treatment. An extreme reduction of the intensity and the size of the fragments have
been observed with the 30%-treated samples as a signal of an extreme degradation.
5.3.3 The influence of acetone treatment
The acetone treatment in the Tutoplast-process aims to remove any residual prions and to
inactivate any enveloped viruses (Schoepf 2006). The acetone wash, followed by vacuum
extraction, dehydrates the tissues to be storable at room temperature.
5. Results Interpretations
102
5.3.3.1 The extent of drying and shrinkage
Figure 5.36 shows that the weight loss, through dehydration, increases almost linearly
with increasing the duration of the acetone series treatment until the tenth day, thereafter
no significant change has been observed. The shrinkage in the volume of the sample is
approximately parallel to the weight loss; it is linear until the tenth day and then it
remained constant.
Time (day)
0 2 4 6 8 10 12 14 16 18 20
Wei
ght l
oss
(%)
0
20
40
60
80
100
Shrin
kage
(%)
0
10
20
30
40
50
60
Figure 5.36: The time-dependence weight loss and volume shrinkage during the
acetone series treatment of native pericardium
In contrast to the acetone series, the loss of the weight is much more quickly by the
treatment with 100% acetone. The samples lose approximately 76% of their weight
within the first 2 days. From the second until the eighteenth day only further 2% weight
is lost, as shown in fig 5.37. Regarding the shrinkage, it behaves differently; it has the
largest shrinkage within the first 2 days, 22.68%, then it increases slowly but significantly
until it reaches a constant value at the sixteenth day, 52.91%.
5. Results Interpretations
103
Time (day)
0 2 4 6 8 10 12 14 16 18 20
Wei
ght l
oss
(%)
0
20
40
60
80
100
Shrin
kage
(%)
0
10
20
30
40
50
60
Figure 5.37: The time-dependence weight loss and volume shrinkage during the
100% acetone treatment of native pericardium
5.3.3.2 The measurements of DC
It is seen obviously that the acetone treatment either 100% or series has no significant
influence on the DC, as shown in fig 5.38. The average DC ± 95 % confidence interval of
the 100%-acetone treated samples is 17.9 % ± 3.2, whereas 15.4 % ± 2.3 for the acetone
series treated samples. The average DC of the control untreated samples is 12.7 % ± 2.1.
5. Results Interpretations
104
DC
(%)
8
10
12
14
16
18
20
22
24
26
w/o series 100 % Figure 5.38: The influence of acetone treatment on the fraction of denatured
collagen, DC, (error bars indicates 95% confidence interval for 6-10
samples)
5.3.3.3 Discussion of the results
Acetone is used in Tutoplast process to inactive the remaining prion and viruses and to
dehydrate the tissues. In the current study the pure acetone was compared with the graded
acetone. After 18 days, both of them have almost the same weight loss and shrinkage,
however with different curve course. The pure acetone is much more effective to
dehydrate the tissues; the maximum weight loss has been achieved within the first two
days almost, whereas the maximum weight loss has been reached at the tenth day during
the graded acetone treatment.
Regarding the shrinkage, during the first 2 days of the pure acetone treatment, the tissue
shrinks 22.68% of their volume. Further shrinkage is also observed until the maximum
shrinkage is reached at the sixteenth day, 52.91%.
5. Results Interpretations
105
The shrinkage of the tissue during the graded acetone treatment is almost parallel to the
weight loss. The shrinkage increases with increasing the treatment duration until it
reached the maximum shrinkage at the tenth day.
During the dehydration process, water is replaced with polar solvents with low H-
bonding abilities, such as acetone, which removes hydrogen bonded water bridges, allows
more direct hydrogen bonding between the molecules and consequently brings the
collagen fibrils closer causing shrinkage (Pashley, Agee et al. 2001; Pashley, Agee et al.
2003; Nalla, Balooch et al. 2005).
The results shows that the pure acetone treatment was more effective to dehydrate the
tissues, however taking into consideration that almost all the water content is removed
within the first two days, it is recommended to stop the pure acetone treatment after the
second day to avoid further shrinkage of the tissue. During the acetone change, the tissues
are taken out from the acetone solution and placed in the air before being weighed and
submerged again in the next acetone solution. In this case, Acetone could be evaporated
from the tissue quickly before being submerged again in the acetone solution. This causes
a gradient in acetone concentration between inside and outside the tissue and
consequently a gradient in the polarity. This could be probable explanation for the further
shrinkage despite the removal of water content.
The functions of the acetone treatment in the Tutoplast-process are tissue dehydration and
inactivation of any prions and viruses. Therefore it is recommended to check if the 2-day
pure acetone treatment is sufficient to inactivate prions and viruses.
The acetone treatment has no significant influence on the collagen denaturation. The DC
values of pure acetone as well as graded acetone treated samples are almost similar to
those of the native untreated samples.
5.4 The mechanical properties of the collagenous tissues
5.4.1 The mechanical properties of bovine bones
The ideal grafting material should not only be adequately osteogenic, -conductive, and -
inductive but also mechanically stable and disease free (Kalfas 2001). Biomechanical
concerns must be carefully considered in the bone healing process during fusion. It has
been appreciated for more than a century that bone forms in places where stress requires
its presence (Pilitsis, Lucas et al. 2002).
5. Results Interpretations
106
The analysis of ultimate strength and elastic modulus values after thermal treatments of
bone cubes, shown in fig 5.39, gives no clear relationship or statement about the
temperature-dependency of the mechanical properties of the bone samples. The 95%
confidence interval for each temperature is very wide as indication of the extreme
scattering of the data. The compressive ultimate strength for the samples treated at 37, 60,
80 and 100 °C are 7.6 ± 1.3, 8.7 ± 3.1, 8.2 ± 2.0 and 10.6 ± 4.2 (mean ± 95% confidence
interval) respectively, whereas the elastic modulus are 25.6 ± 8.8, 42.6 ± 21.2, 38.5 ±
13.2 and 51.9 ± 26.4 respectively.
37 °C 60 °C 80 °C 100 °C 37 °C 60 °C 80 °C 100 °C
Elas
tic M
odul
us (M
Pa)
0
20
40
60
80
100
120
Ulti
mat
e St
reng
th (M
Pa)
0
5
10
15
20
Figure 5.39: The temperature-dependence mechanical properties of bovine
cancellous bone (error bars indicate 95 % confidence interval of 9
samples)
5.4.2 The mechanical properties of bovine pericardium
The mechanical properties of the pericardium reflect the ‘the healthiness’ and the
structural integrity. Any modification or destruction induced by processing should be
detected in a biomechanical analysis.
5. Results Interpretations
107
As observed with the bone samples, Figure 5.40 does not show any reasonable
relationship or conclusion about the influence of the processing on the mechanical
stability of the pericardium samples. Wide 95% confidence intervals have been observed,
which prevent any assessment of the effect of the processing. The tensile ultimate
strength for the left side native and processed, and the right side native and processed
were (mean ± 95% confidence interval) 9.2 ± 4.4, 12.5 ± 3.5, 8.9 ± 8.9 and 6.8 ± 1.7
respectively, whereas the elastic modulus were 113.5 ± 53.4, 115.6 ± 52.8, 93.0 ± 87.9
and 80.8 ± 24.5 respectively.
Ulti
mat
e ST
reng
th (M
pa)
-5
0
5
10
15
20
Elas
tic M
odul
us (M
Pa)
0
50
100
150
200
N P
N P
N P
N P
Left
Left
Right
Right Figure 5.40: The influence of Tutoplast process on the mechanical properties of
bovine pericardium (error bars indicate 95 % confidence interval of 5
samples)
5.4.3 Discussion of the results
The bone has several functions in the body but at least two key biomechanical roles (Burr
and Turner 2003); bones shield vital organs from trauma and serve as levers against
5. Results Interpretations
108
which muscles contact. Bone is a natural composite, in which Minerals are responsible
for the bone stiffness and collagen for the bone toughness (Wang, Bank et al. 2001; Burr
and Turner 2003).
The bone samples were heated in the range 37-100 °C before being tested to induce
collagen denaturation, which supposed to be important in determining the mechanical
properties of the bones. The results do not show any relationship between the temperature
and the mechanical properties. Wang et al (Wang, Bank et al. 2001) heated cortical
samples in the range 37-200 °C before being tested in three-point bending configuration.
They showed that heating induces denaturation, which lead to a significant decrease in
the toughness of bone but has little effect on the stiffness of the bone. The deviation of
the current results from those of Wang et al could be attributed to different reasons;
Wang et al didn’t observe any influence of the thermal denaturation on the mechanical
properties up to 160 °C. Furthermore they used the three-point bending configuration,
which combination between tension and compression (Einhorn 1992), whereas in this
study compression test was used.
Assuming that heating in the range used in this study has no influence on the mechanical
properties, the mechanical properties of all the samples should be almost identical
however, it is not the case and the results are scattered.
The influence of the structure heterogeneity and the fiber orientation could be behind the
scattering of the results and the absence of convenient statement about the mechanical
properties. Fratzl et al (Fratzl, Gupta et al. 2004) suggested that the bone matrix is not
uniformly mineralized, but shows a pronounced local variation. Therefore the bone
material is composed of bone packets, each having its own mineral content corresponding
to its tissue age. In the current work, taking the samples without previous knowledge of
the mineral content could be a reason for the scattering of the results.
Furthermore, Peterlik et al (Peterlik, Roschger et al. 2006) found that the fracture energy
changes by two orders of magnitude depending on the collagen orientation, and the angle
between collagen and crack propagation direction is decisive in switching between
different toughening mechanisms. In the current study 1×1×1 cm3 cubes were cut from
the same direction, however the confusion of the orientation during the thermal treatment
5. Results Interpretations
109
and then during the mechanical test as experimental error is not excluded, which may
contribute in the scattering of the results.
The pericardium is a viscoelastic material (Paez and Jorge-Herrero 1999), i.e. the stress
strain relationship is non linear. Therefore the Hooke’s law is normally not considered for
the analysis of the mechanical properties of the pericardium. Despite this fact, Hooke’s
law has been used to describe the linear region of the stress strain diagram (Zioupos and
Barbenel 1994). In this study, it was assumed that Hooke’s law is valid in the linear
region of the stress stain diagram.
It is known in the literature that Bovine pericardium is generally considered to be
mechanically anisotropic (Lee, Courtman et al. 1984; Zioupos and Barbenel 1994). To
overcome the mechanical anisotropy of the pericardium, some researchers use the small
angle light scattering (SALS) to quantify the collagen fiber architecture and to select
samples with minimum variability to be used in the mechanical tests (Hiester and Sacks
1998; Hiester and Sacks 1998; Sacks and Chuong 1998; Mirnajafi, Raymer et al. 2005).
Others separated between the left and the right side of the pericardial sac (Garcia Paez,
Jorge-Herrero et al. 2001). In the current work, native as well as Tutoplast-processed
samples from the left and the right sides of the pericardium were taken for the analysis of
the mechanical properties.
The analysis of the results does not lead to any conclusion about the influence of the
processing of the mechanical stability. It is expected, despite the separation between the
left and the right side of the sac, that the effect of anisotropy was dominant over the
influence of the processing. It can be concluded that only SALS-selected samples can be
used to assess the effect of the processing on the mechanical properties of the
pericardium.
6. Optimization of Tutoplast Process
110
6 Optimization of Tutoplast process The optimization of the process aims to achieve one or more of the following goals
• Improving the quality of the processed tissues
• Shortening the process duration
• Reduction of the processing costs.
Any kind of optimization that saves time and costs with worsening the quality of the
tissues will not be taken into consideration.
The effect of Tutoplast processing of the stability of bovine pericardium was studied. It
was concluded that the processing induces structural destruction. The contribution of
each step in the process was followed; analyzed and practical solutions were suggested as
the following:
6.1 The sodium hydroxide treatment Tutoplast process
During Tutoplast process, the tissues are treated with 1 N NaOH for 1 h. This treatment
induces osmotic swelling of the tissues and reduction of the thermal shrinkage. The
action of alkali on the collagen is represented by amino acids modification and
destruction of intra-and intermolecular collagen cross-links.
In order to restore the tissues to their physiological state, the tissues are treated with 1 N
CH3COOH for 15 min. This treatment shifts the pH value of the strips too rapidly from
the basic to the acidic region (from 14 to 3) causing swelling and lower thermal stability
also.
After the 15-min 1 N CH3COOH, the tissues are submerged in RO-water for 30 min,
which was found inefficient to restore the tissues to their physiological state.
Alternatives
The 1 N NaOH treatment for 1 h is a validated step against CJD, therefore it was kept
unchanged. As alternatives to the 15-min 1 N CH3COOH as neutralization step, the 15
min 0.1 N CH3COOH was more efficient and yielded tissues with pH 6 and thermal
shrinkage close to that of the native untreated tissues.
6. Optimization of Tutoplast Process
111
Restoring the tissues to their physiological state after the NaOH treatment by washing
with water bath is possible but it takes long time, at least 90 min, with changing the water
bath every 10-15 min.
The best variant was to treat the NaOH-treated strip with 0.1 N CH3COOH for 15 min
followed by one or two 10-min water baths.
6.2 The hydrogen peroxide treatment Tutoplast
The tissues are treated during the process with 3 % H2O2 for 72 h. This step induces little
reduction of the thermal shrinkage of the strips.
Alternatives
Using 10 % H2O2 could be more effective to oxidize the proteins and to inactivate the
viruses. It was seen that the 10 % H2O2 treatment results in tissues have almost the
similar shrinkage temperature as that of the 3 % H2O2 treated tissues. Therefore using the
10 % will almost not induce further destruction
6.3 The acetone treatment Tutoplast
During the process the tissues are treated with pure acetone for two weeks. This steps
aims to dehydrate the tissues and inactivate the remaining prions
Alternatives
The pure acetone used in the process was found to be more efficient than graded acetone
as dehydrating agent. However, the 2-week treatment is too long and caused avoidable
shrinkage. It was found that after 2 days the tissues were fully dehydrated.
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1 Introduction................................................................................................................. 1 2 State of the art ............................................................................................................. 2
2.1 Bone grafting ...................................................................................................... 2 2.1.1 Graft types................................................................................................... 2
2.1.1.1 Autograft ................................................................................................. 2 2.1.1.2 Allograft.................................................................................................. 2 2.1.1.3 Xenograft ................................................................................................ 3 2.1.1.4 Alloplastic ............................................................................................... 3
2.1.2 Bone healing ............................................................................................... 4 2.1.3 Graft processing .......................................................................................... 6
2.1.3.1 Graft processing techniques used in the medical field............................ 6 2.1.3.2 Tutoplast® process ................................................................................. 7
2.2 Collagen ............................................................................................................ 10 2.2.1 Collagen types........................................................................................... 10 2.2.2 Collagen synthesis .................................................................................... 12 2.2.3 Collagen cross-links.................................................................................. 14 2.2.4 The role of hydroxyproline in collagen stabilization................................ 16 2.2.5 The thermal stability of collagen .............................................................. 18 2.2.6 Advanced glycation end products (AGEs) ............................................... 19
2.3 Bone .................................................................................................................. 20 2.3.1 Bone composition ..................................................................................... 20 2.3.2 Bone hierarchy .......................................................................................... 21
2.4 Pericardium....................................................................................................... 23 3 The Objectives .......................................................................................................... 26 4 Materials and Methods.............................................................................................. 27
4.1 Materials ........................................................................................................... 27 4.1.1 Bovine Bones ............................................................................................ 27 4.1.2 Bovine Pericardium .................................................................................. 28
4.2 Methods............................................................................................................. 29 4.2.1 Preparation Steps ...................................................................................... 29
4.2.1.1 The pulverization of the bones.............................................................. 29 4.2.1.2 The Demineralization of Bones ............................................................ 30
4.2.2 The Determination of Denatured Collagen (DC)...................................... 30 4.2.2.1 A Selective Digestion Method .............................................................. 31 4.2.2.2 Spectrophotometeric Determination of the DC .................................... 32
4.2.3 The Measurements of the Extent of Browning ......................................... 34 4.2.4 The Measurements of the Isotonic Shrinkage temperature....................... 34 4.2.5 SDS-PAGE ............................................................................................... 35 4.2.6 Characterization of the Mechanical Properties ......................................... 39 4.2.7 The measurements of the Thermal Conductivity...................................... 39
4.3 Different physical and chemical treatment ....................................................... 39 4.3.1 The thermal treatment of collagenous tissues........................................... 40
4.3.1.1 The thermal treatment of bovine bone .................................................. 40 4.3.1.2 The thermal stability of bovine pericardium......................................... 40
4.3.2 The Sodium hydroxide treatment and the corresponding neutralization .. 41
4.3.2.1 Sodium hydroxide treatment................................................................. 41 4.3.2.2 The neutralization of the tissues after the NaOH treatment.................. 42
4.3.3 The hydrogen peroxide treatment ............................................................. 43 4.3.4 The acetone treatment ............................................................................... 43 4.3.5 The determination of water content .......................................................... 44
5 Results Interpretations .............................................................................................. 45 5.1 The effect of bone pulverization on the collagen.............................................. 45
5.1.1 The measurements of DC.......................................................................... 45 5.1.2 Discussion of the results ........................................................................... 46
5.2 The thermal stability of collagenous tissues ..................................................... 47 5.2.1 Analysis of the thermal stability with the measurements of DC .............. 47
5.2.1.1 The thermal stability of Tutoplast-processed bovine bone ................... 47 5.2.1.2 The thermal stability of native bovine pericardium.............................. 48 5.2.1.3 The thermal stability of tutoplast-processed bovine pericardium......... 49 5.2.1.4 The thermal stability of the lyophilized bovine pericardium................ 50 5.2.1.5 The measurements of the extent of browning....................................... 51 5.2.1.6 Discussion of the results ....................................................................... 52 5.2.1.7 Modeling of the thermal denaturation of pericardium.......................... 59
5.2.2 Measurements of isotonic shrinkage temperature..................................... 65 5.2.2.1 The results............................................................................................. 66 5.2.2.2 Discussion of the results ....................................................................... 66 5.2.2.3 Modelling of the thermal shrinkage of pericardium ............................. 68
5.2.3 SDS-PAGE investigations ........................................................................ 78 5.2.3.1 Results................................................................................................... 78 5.2.3.2 Discussion ............................................................................................. 81
5.3 The effect of different steps in the Tutoplast® process .................................... 83 5.3.1 The effect of sodium hydroxide treatment................................................ 83
5.3.1.1 The hydrolysis of collagen amino acids................................................ 84 5.3.1.2 The measurements of the shrinkage temperature.................................. 86
5.3.1.2.1 The effect of NaOH solution........................................................... 86 5.3.1.2.2 The effect of the neutralization step................................................ 87
5.3.1.3 The measurements of DC...................................................................... 91 5.3.1.4 SDS-PAGE investigations .................................................................... 92 5.3.1.5 Discussions ........................................................................................... 94
5.3.2 The effect of hydrogen peroxide treatment............................................... 98 5.3.2.1 The shrinkage temperature measurements............................................ 98 5.3.2.2 Measurements of DC ............................................................................ 98 5.3.2.3 SDS-PAGE investigations .................................................................... 99 5.3.2.4 Discussion of the results ..................................................................... 100
5.3.3 The influence of acetone treatment......................................................... 101 5.3.3.1 The extent of drying and shrinkage .................................................... 102 5.3.3.2 The measurements of DC.................................................................... 103 5.3.3.3 Discussion of the results ..................................................................... 104
5.4 The mechanical properties of the collagenous tissues .................................... 105 5.4.1 The mechanical properties of bovine bones............................................ 105 5.4.2 The mechanical properties of bovine pericardium.................................. 106
5.4.3 Discussion of the results ......................................................................... 107 6 Optimization of Tutoplast process .......................................................................... 110
6.1 The sodium hydroxide treatment .................................................................... 110 6.2 The hydrogen peroxide treatment ................................................................... 111 6.3 The acetone treatment ..................................................................................... 111
7 Literature................................................................................................................. 112
Einleitung Verschiedene Transplantationsmaterialien, einschließlich Autografts, Allografts,
Xenografts und synthetische Materialien, im klinischen Bereich werden eingesetzt. Das
Autograft wird als das Goldstandardtransplantat betrachtet, weil es intakte Zellen und
Wachstumsfaktoren enthält, welche die Heilung des Transplantates anregen. Jedoch sind
die begrenzte Verfügbarkeit und die zusätzliche Morbidität die Hauptnachteile, die mit
der Verwendung von Autografts verbunden sind. Deshalb müssen alternative
Transplantationsmaterialien verwendet werden, um die zunehmende Nachfrage nach
Transplantaten im medizinischen Bereich zu erfüllen.
Allografts werden zwar häufig eingesetzt aber das Risiko immunologischer Reaktionen
und die Übertragung von Krankheiten schränken deren Verwendung ein.
Die Risiken bei der Verwendung von Allografts und Xenografts (Antigenität und
Infektionsgefahr) können durch verschiedene Behandlungen verringert werden. Leider
führen solche Behandlungen oft zu Veränderungen der mechanischen und biologischen
Gewebeeigenschaften.
Der Tutoplast® Prozess stellt einen kompletten und validierten Konservierungs- und
Sterilisationsprozess, der seit 30 Jahren verwendet wird. Dieser Prozess zielt auf saubere
und sichere Transplantate durch die Beseitigung von Antigenität sowie viralen
Krankheiten, ohne die mechanischen und biologischen Eigenschaften der Gewebe zu
verändern. Der Tutoplast® Prozess deaktiviert, zerstört und entfernt alle unerwünschten
Materialien aus den prozessierten Geweben, wie Fette, Zellen, Viren und Mikroben.
In der hier vorliegenden Dissertation wird der Einfluss des Tutoplast® Prozesses und die
Auswirkung jeder seiner Einzelschritte auf die thermischen und mechanischen
Eigenschaften von Knochen und Perikard beurteilt. Dies stellt die Grundlage für die
zukünftigen Pläne einer Prozessoptimierung dar.
Zielsetzung Die Nachfrage nach den biologischen Implantaten steigt stetig an. Um die zunehmende
Marktnachfrage zu erfüllen, muss die Produktion an Implantaten in naher Zukunft erhöht
werden. Diese Tatsachen weisen zwingend auf eine notwendige Optimierung des
Tutoplast Prozesses hin, ohne dabei die Produktqualität (prozessierte Gewebe) zu
verschlechtern.
Für eine Prozessoptimierung bedarf es einer vollständigen Überprüfung und Analyse
eines jeden Prozesseinzelschrittes. Dies stellt die Basis für künftige
Optimierungsversuche dar.
Zuerst wird der Einfluss des Tutoplast Prozesses auf die thermische Stabilität der
prozessierten Knochen und Perikard untersucht, da die thermische Stabilität ein guter
Hinweis auf die Unversehrtheit der Gewebe ist. Weiterhin ist die Kenntnis der
thermischen Stabilität wichtig, um den Einfluss einer thermischen Behandlung der
Gewebe während medizinischer Therapien zu verstehen.
Native und prozessierte Perikardproben werden thermisch behandelt und anschließend
enzymatisch verdaut, um den Anteil an denaturiertem Kollagen (DC) zu bestimmen. Zur
Überprüfung der jeweiligen Prozesseinzelschritte werden native Proben separat mit
NaOH, H2O2 und Aceton bei unterschiedlichen Bedingungen (Konzentration,
Einwirkdauer) behandelt und anhand von Messungen der isotonischen
Schrumpfungstemperatur sowie DC Messungen und SDS-PAGE bewertet.
Der Einfluss des Tutoplast-Prozesses auf die mechanischen Eigenschaften der
prozessierten Knochen und Perikard wird durch Druck- und Zugfestigkeitsversuche an
entsprechenden Proben ermittelt.
Zusammenfassung Die hier vorliegende Arbeit konzentriert sich auf das Aufzeigen von
Optimierungsmöglichkeiten für den Tutoplast® Prozess. Die Optimierungsversuche
wurden durch unterschiedliche Qualitätssicherungstests evaluiert. Um eine
Prozessoptimierung des Prozesses durchzuführen, musste zunächst der Einfluss des
Prozesses auf die Materialien Knochen und Perikard sorgfältig definiert werden.
Weiterhin war der Einfluss eines jeden Einzelschrittes separat zu untersuchen und durch
die gültigen Qualitätssicherungstests zu beurteilen.
Die thermische Stabilität der Kollagengewebe wurde als entscheidender Parameter
betrachtet. Die Perikardproben wurden im Bereich von 55 bis 200 °C für 1 h im
Trockenschrank thermisch behandelt und anschließend mit α-Chymotrypsin verdaut, um
den Anteil an denaturiertem Kollagen (DC) zu bestimmen. Die DC Messungen zeigten
bei nativen lyophilisierten (Wassergehalt 7%) und Tutoplast-prozessierten Perikard
(Wassergehalt 1.7%) eine höhere thermische Stabilität, im Vergleich zu nativem Perikard
(Wassergehalt 85%). Der DC Anteil für das native lyophilisierte und Tutoplast-
prozessierte Perikard blieb bis 135 °C unverändert, während sich der DC Anteil bei
nativem Perikard bereits ab 55 °C zu erhöhen begann. Dies konnte auf den Wassergehalt
im Perikard zurückgeführt werden. Nach dem Polymer in a Box Mechanismus, begrenzt
eine Dehydratisierung die Anzahl möglicher Konfigurationen, verringert damit die
Konfigurationsentropie und erhöht dadurch die thermische Stabilität des Kollagens.
Der Einfluss des Wassergehalts während der DC Messungen wurde dadurch eliminiert,
dass die Schrumpfungstemperaturmessungen in einem Wasserbad durchgeführt werden.
durch Schrumpfungstemperaturmessungen lassen sich Destruktionen oder Änderungen in
der Gewebestruktur zu erkennen.
Es wurde festgestellt, dass das Tutoplast-prozessierte Perikard eine niedrigere
Schrumpfungstemperatur aufweist. Ein Hinweis auf strukturelle Gewebeveränderungen
bzw. Zerstörungen, welche durch die Prozessierung verursacht werden. Tutoplast-
prozessiertes Perikard beginnt ab 42 °C zu schrumpfen, während der Schrumpfprozess
bei nativem lyophilisiertem und nativem Perikard ab 64 bzw. 65 °C einsetzt.
Die Ergebnisse aus den DC- und Schrumpfungstemperaturmessungen führen zu der
Schlussfolgerung, dass der Tutoplast-Prozess strukturelle Gewebeänderungen induziert,
die unter trockenen Bedingungen nicht nachweisbar sind. Nachfolgend wurde der Beitrag
eines jeden Prozesseinzelschrittes auf die strukturelle Gewebeänderung untersucht.
Hinsichtlich der Natriumhydroxidbehandlung wurde festgestellt, dass 1 N NaOH eine
Modifizierung der Aminosäuren, und eine erhebliche Senkung der
Schrumpfungstemperatur verursacht. Des Weiteren wurde nachgewiesen, dass eine
NaOH Behandlung bei Raumtemperatur zu einer Hydrolyse des Kollagens führt, die
jedoch als nicht signifikant eingestuft werden kann.
Um den Einfluss der NaOH Behandlung zu eliminieren, wurden einige
Neutralisationsmöglichkeiten geprüft. Als beste Variante stellte sich eine 15-minütige
Behandlung der Proben mit 0,1 N CH3COOH und zwei anschließenden 10-minütigen
Wasserspülungen dar.
Im Fall der Wasserstoffperoxidbehandlung (H2O2) wurde nachgewiesen, dass eine 48-
stündige der Gewebe Behandlung mit 3 und 10% H2O2 Lösungen kaum zu nachteiligen
Gewebeveränderungen führt. Die Schrumpfungstemperaturen der so behandelten Proben
lagen um 5 bzw. 7 Grad niedriger als die der unbehandelten Proben. Die Behandlung mit
30% H2O2 Lösung war extrem destruktiv, so dass die Proben für weitere Messungen nicht
mehr herangezogen werden konnten.
Hinsichtlich des Acetondehydratisierungschrittes wurde gezeigt, dass eine Behandlung in
reinem Aceton einen effektiveren Prozessschritt darstellt als eine aufsteigende
Acetonreihe. Jedoch führt die lange Behandlungsdauer zu unerwünschter Schrumpfung.
Die DC- Messungen zeigten in beiden Fällen (reines Acetons/Acetonreihe) keinen
Einfluss auf die Stabilität des Kollagens.
Die mechanischen Eigenschaften der Knochen und Perikard wurden ebenfalls
charakterisiert. Im Temperaturbereich von 37 bis 100 °C konnte keine signifikante
Abhängigkeit der Druckfestigkeit boviner Knochen nachgewiesen werden. Der Grund
hierfür könnte in der strukturellen Heterogenität und der Faserorientierung dieses
Materials liegen.
Ein Einfluss des Tutoplast-Prozesses auf die Zugfestigkeit des Perikards konnte nicht
nachgewiesen werden. Hier könnte der Grund in der mechanischen Anisotropie des
Materials liegen.