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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• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 1 Mo

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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.