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Characterization and development of optimization strategy for the processing of allogenic and xenogenic bone and pericardium [Elektronische Ressource] / presented by Mohannad Qasim Mustafa Marashdeh

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149 pages
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.
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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
COOH is not suitable to reversible. It was observed that treating the tissues with 1 N CH3
restore the tissues to their physiological state. The best variant was to treat the tissues
with 0.1 N CH COOH followed by 1-2 10-min water baths. 3
The 3 % H O had little effect on the quality of the tissues. Furthermore, 10 % H O 2 2 2 2
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 CH COOH 3
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 CH COOH treatment 3
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 CH COOH. The 3
15-min treatment with 0.1 N CH COOH shifted the pH value of the pericardium strips to 3
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
CH COOH for 15 min followed by one or two 10-min distilled water washing bath. With 3
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