PhD THESIS

171 Pages·2016·5.32 MB·English

PhD THESIS

"INVESTIGATION OF THE EFFECT LASER IRRADIATION ON
SURFACE STRUCTURE OF CERAMIC MATERIALS"

«ΔΙΕΡΕΥΝΗΣΗ ΤΗΣ ΕΠΙΔΡΑΣΗΣ ΑΚΤΙΝΩΝ LASER ΣΤΗ ΔΟΜΗ
ΤΗΣ ΕΠΙΦΑΝΕΙΑΣ ΚΕΡΑΜΙΚΩΝ ΥΛΙΚΩΝ»

Anastasia Beketova

Aristotle University of Thessaloniki, School of Dentistry
Department of Fixed Prosthesis and Implant Prosthodontics
Head of the Department: Professor P. Koidis

Thessaloniki, 2016
Investigation of the Effect of Laser Irradiation on
Surface Structure of Ceramic Materials

PhD Thesis

Anastasia Beketova,
dentist

Supervisor:

Dr. Petros Koidis, Professor,
Head of the Department of Fixed Prosthesis and Implant Prosthodontics,
Faculty of Dentistry, School of Health Sciences Aristotle University of
Thessaloniki.

Co-supervisors:

Dr. Konstantinos M. Paraskevopoulos, Professor,
Physics Department, Aristotle University of Thessaloniki

Dr. Gerasimos Kourouklis, Professor,
Department of Chemical Engineering, School of Engineering, Aristotle
University of Thessaloniki

Examining Committee:

Dr. Nikolaos Poulakis, Professor,
Department of Electrical Engineering, Technological Educational Institute of
Western Macedonia, Koila, Kozani

Dr. Argiris Pisiotis, Professor,
Department of Removable Prosthodontics, Faculty of Dentistry, School of
Health Sciences Aristotle University of Thessaloniki

Dr. Eleana Kontonasaki, Assistant Professor,
Department of Fixed Prosthesis and Implant Prosthodontics, Faculty of
Dentistry, School of Health Sciences Aristotle University of Thessaloniki

Dr. Athina Bakopoulou, Assistant Professor,
Department of Fixed Prosthesis and Implant Prosthodontics, Faculty of
Dentistry, School of Health Sciences Aristotle University of Thessaloniki
Dedicated to my beloved family
Table of Contents

Acknowledgements ........................................................................................................... 6
Abstract ............................................................................................................................ 7
Περίληψη .......................................................................................................................... 8
Introduction and Skope ....................................................................................................10
1. Chapter: Literature review .........................................................................................13
1.1 Bioceramics .......................................................................................................13
1.1.1 Bioceramics Classification ..........................................................................13
1.1.2 Dental Feldspathic Porcelain ......................................................................14
1.1.3 Bioactive Glasses .......................................................................................16
1.1.4 Bioactive glass-Dental ceramic composites ................................................19
1.2 Calcium Orthophosphates .................................................................................21
1.2.1 Hydroxyapatite Structure ............................................................................23
1.2.2 Biological Hydroxyapatite............................................................................25
1.2.3 Crystallization of Calcium Phosphates from Supersaturated Solutions .......28
1.3 Structure of Dental Cementum...........................................................................30
1.4 Laser .................................................................................................................33
1.4.1 Interaction of Laser Light with Ceramic Materials ........................................34
1.5 Methods of Materials Characterization ...............................................................40
2 Chapter: Materials and Methods ...............................................................................43
2.1 Synthesis, characterization of BP80 and BP67 bioceramic materials and
evaluation of their apatite forming ability .......................................................................44
2.1.1 Synthesis of BP80 and BP67 bioceramic materials by sol-gel method ........44
2.1.2 Fabrication of sintered specimens ..............................................................47
2.1.3 Characterization of the fabricated composite materials ...............................47
2.1.4 Evaluation of the apatite forming ability of BP80 and BP67 composites ......48
2.1.5 Characterization of the morphology and chemical composition of the BP67
and BP80 sample’s surface after immersion to 1.5xSBF ...........................................50
2.2 Development of an appropriate experimental set-up for Laser Assisted
Biomimetic- LAB process (pilot studies) .......................................................................50
2.2.1 Description of the LAB method ...................................................................50
2.2.2 Overview of the pilot studies .......................................................................51
2.3 Evaluation of the optimum laser irradiation parameters for BP67 material .........52
2.3.1 Experimental Set-Up for LAB ......................................................................52
2.3.2 Irradiation of BP67 with different laser light densities (fluence) ...................55
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2.3.3 Irradiation of the BP67 specimens for different time periods .......................55
2.4 Investigation of the chemical composition, morphology of the obtained HA
coating and cell vability/proliferation studies .................................................................56
2.4.1 Characterization of the deposited coatings by micro Raman and SEM in
cross-sections ...........................................................................................................56
2.4.2 Cell culture studies .....................................................................................57
3 Chapter: Results .......................................................................................................61
3.1 Structural characterization of BP80 and BP67 materials and evaluation of their
apatite forming ability ...................................................................................................61
3.1.1 Characterization of BP67 and BP80 materials by FTIR and XRD ...............61
3.1.2 Evaluation of the apatite forming ability of BP67 and BP80 materials .........63
3.2 Investigation of different experimental conditions applied to BP80 composite,
pilot studies (See Annexes) .........................................................................................68
3.3 Evaluation of the optimum laser irradiation parameters for BP67 material .........68
3.3.1 Irradiation of BP67 with different laser light densities (fluencies) .................68
3.3.2 Irradiation of the BP67 specimens for different time periods .......................70
3.4 Investigation of the chemical composition, morphology of the obtained HA
coating and cell vability/proliferation studies .................................................................75
3.4.1 Characterization of the deposited coatings by micro Raman and SEM in
cross-sections ...........................................................................................................75
3.4.2 Cell culture studies .....................................................................................77
4 Chapter: Discussion ..................................................................................................85
5 Conclusions ..............................................................................................................97
6 Annexes ...................................................................................................................98
6.1 Annex 1. Application of different parameters of Nd:YAG laser light (1064nm) to
BP80 composite during LAB .........................................................................................98
6.1.1 Materials and Methods ...............................................................................98
6.1.2 Results ..................................................................................................... 100
6.1.3 Conclusions .............................................................................................. 103
6.2 Annex 2: Irradiation of BP80 material by Nd:YAG (355nm) laser in cSBF solution
during LAB ................................................................................................................. 104
6.2.1 Materials and Methods ............................................................................. 104
6.2.2 Results ..................................................................................................... 106
6.2.3 Conclusions .............................................................................................. 108
6.3 Annex 3: Irradiation of BP80 material by KrB laser (248nm) in cSBF during LAB
109
6.3.1 Materials and Methods ............................................................................. 109
6.3.2 Results ..................................................................................................... 110
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6.3.3 Conclusions .............................................................................................. 113
6.4 Annex 4: Spectrophotometric measurements of reflectivity of BP80 material and
absorbance of cSBF solution ...................................................................................... 114
6.4.1 Materials and Methods ............................................................................. 114
6.4.2 Results ..................................................................................................... 114
7 References ............................................................................................................. 116

Εκτεταμένη Ελληνική Περίληψη………………………………………………........ 143

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Acknowledgements

I would like to express my deep and sincere gratitude to my supervisor, Professor P. Koidis,
Head of the Department of Fixed Prosthesis and Implant Prosthodontics for his support though out
my thesis, his endless patience and knowledge. Under his guidance during all these years I grew
up as a person, and I attribute my PhD degree to his encouragement, effort and trust in me. My
very special thanks to my co-supervisors, Professor Paraskevopoulos and Professor Kourouklis,
for their scientific guidance and precious advice in every single step of the research and given
research facilities. It is difficult to overestimate my gratitude to Dr. Poulakis, Professor in the
Department of Electrical Engineering, Technological Educational Institute of Western Macedonia
for his enormous contribution to this PhD thesis, without his help it wouldn’t be possible to perform
the experiments with laser. I am also very thankful for his professionalism, kindness and support.
I would like to express my gratitude to Dr. Bakopoulou for her help in experiments with cell
cultures, her enthusiasm was very motivational for me. I am heartily thankful to Dr. Zorba and Dr.
Papadopoulou and Dr. Kontonasaki who contributed with their experience and work to the
realization of this thesis and also for their kindness and encouragement. My special thanks to Dr.
Christofilos for help and guidance through the development of my thesis. I also would like to
acknowledge Dr. Kantiranis for XRD analysis and Professor Zachariadis for ICP analysis.
A part of experiments with laser were performed also in Crete, therefore I would like to
acknowledge Professor Anglos, Dr. Klini, Dr. Papoutsakis for their contribution to this PhD thesis.
I would like to thank G. Theodorou for his help with experiments and all the members of the
FTIR group, who supported me in difficult moments. Lots of thanks to my family, especially to my
parents and grandparents, for their emotional, moral, and financial support and to my friends also.
This PhD dissertation was financed by several institutions and foundations. I would like to
express my sincere gratitude to the Greek State Scholarship Foundation (IKY) that granted me with
a scholarship for the years 2012-2015, the Research Committee of Aristotle University of
Thessaloniki that granted me with a scholarship for the year 2011 and Erasmus Mundus
Cooperation Window Lot 7 for the necessary financial support for this research in 2010-2011.
Special thanks to my academic supervisor Professor Koidis and Professor Paraskevopoulos for
their contributions towards my scholarships.
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Abstract

Objectives: Aims of this study were to investigate the optimal conditions of laser irradiation
of Bioactive Glass (B) /Dental Porcelain (P) BP80 a novel BP67 composites for acceleration of
hydroxyapatite-HA formation and to assess cellular responses on the precipitated HA region.
Materials and Methods: BP80 (B:20%, P:80%) and BP67 (B:33.3%, P:66.7%) composites
were fabricated by the sol-gel method, and their composition and apatite forming ability was
evaluated. Laser assisted biomimetic (LAB) process was applied to both materials. The effect of
various energy densities of and irradiation exposure times were evaluated for HA precipitation. The
obtained HA film was characterized by FTIR, XRD, SEM and micro Raman techniques. ICP-AES
was used for revealing changes in chemical composition of the precursor liquid during irradiation.
Cell viability and morphological characteristics of periodontal ligament fibroblasts-PDLFs, human
gingival fibroblasts-HGFs and SAOS-2 osteoblasts on the HA surface were evaluated by MTT
assays and SEM.
Results and Discussion: Laser irradiation of BP80 material under the applied conditions
did not cause accelerated precipitation of HA due to low bioactivity of the material. Laser treatment
of BP67 at optimal energy fluence of 1.52 J/cm2 and irradiation time for 3 h followed by immersion
in 1.5xSBF at 60oC, resulted in formation of a dense HA layer within 7 days instead of 25 d
required for HA precipitation during conventional biomimetic process. The accelerated formation of
HA film during LAB could be attributed to increase of material’s solubility in the laser irradiated
spot, together with increase of thermodynamic energy of the ion species and supersaturation in the
solution. The resulting HA film was tightly bonded to the underlying substrate and had mineral
composition similar to dental cementum. MTT assay showed a consistent reduction of cell
proliferation on the HA layer in comparison to conventional control ceramic and BP67 for all 3 cell
lines studied.
Conclusion: LAB process is potentially applicable for functionalization of materials with low
bioactivity under precise control of laser irradiation parameters, while cellular responses need
further investigation.

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Περίληψη

Σκόπος: Ο στόχος αυτής της μελέτης ήταν η διερεύνηση των βέλτιστων συνθηκών ακτινοβόλησης
με λέιζερ δύο σύνθετων βιοκεραμικών υλικών, των BP80 και BP67 συστημάτων οδοντιατρικής
πορσελάνης (P) και βιοενεργού υάλου (B), για την επιτάχυνση σχηματισμού υδροξυαπατίτη (HA)
στην επιφάνια αυτών, καθώς και την αξιολόγηση των κυτταρικών αποκρίσεών τους στην περιοχή
του ΗΑ.
Υλικά και Μέθοδοι: Τα σύνθετα υλικά BP80 (Β: 20%, P: 80%) και BP67 (Β: 33,3%, Ρ: 66,7%)
κατασκευάστηκαν με την μέθοδο sol-gel, και αξιολογήθηκαν η σύνθεσή τους και η ικανότητά αυτών
για το σχηματισμό απατίτη. Η μέθοδος LAB (Laser Assisted Biomimetic) εφαρμόστηκε και στα δύο
υλικά. Η επίδραση των διαφόρων ενεργειακών πυκνότητων ακτινοβολίας και χρόνου έκθεσης
αξιολογήθηκαν για την ανάπτυξη του ΗΑ. Το ληφθέν φιλμ του ΗΑ χαρακτηρίστηκε με τις τεχνικές
FTIR, XRD, SEM και micro Raman. Επιπλέον, η φασματοσκοπία ICP-AES χρησιμοποιήθηκε για
την ανίχνευση αλλαγών στη χημική σύνθεση του βιομιμητικού διαλύματος κατά τη διάρκεια της
ακτινοβόλησης. Η βιωσιμότητα καθώς και τα μορφολογικά χαρακτηριστικά των περιοδοντικών
ινοβλαστών PDLFs, των ουλικών ινοβλαστών HGFs και των οστεοβλαστών SAOS-2 στην
επιφάνεια ΗΑ αξιολογήθηκαν με την δοκιμασία ΜΤΤ και με ηλεκτρονική μικροσκοπία SEM.
Αποτελέσματα και Συζήτηση: Η ακτινοβόληση με λέιζερ του σύνθετου υλικού BP80 υπό τις
εφαρμοζόμενες συνθήκες δεν προκάλεσε την επιταχυνόμενη ανάπτυξη του ΗΑ, γεγονός το οποίο
οφείλεται στη μη βιοενεργή συμπεριφορά αυτού του υλικού. Η αγωγή με λέιζερ του σύνθετου
υλικού BP67 στη βέλτιστη ενεργειακή ροή των 1,52 J/cm2 και για χρόνο ακτινοβόλησης τριών
ωρών, ακολουθούμενη από διατήρηση των δοκιμίων σε διάλυμα 1.5x SBF στους 60°C, είχε ως
αποτέλεσμα το σχηματισμό ενός πυκνού στρώματος ΗΑ μέσα σε 7 ημέρες αντί για 25 ημέρες που
απαιτούνται για την καθίζηση του ΗΑ σύμφωνα με τη συμβατική βιομιμητική μέθοδο (conventional
biomimetic method). Ο επιταχυνόμενος σχηματισμός του υμενίου από απατίτη κατά τη διάρκεια της
LAB θα μπορούσε να αποδοθεί σε αύξηση της διαλυτότητας του υλικού στη περιοχή
ακτινοβόλησης με λέιζερ, και στην αύξηση της θερμικής ενέργειας των ιόντων καθώς και λόγω
υπερκορεσμού του διαλύματος. Το σχηματισθέν στρώμα του ΗΑ ήταν καλά συνδεδεμένο με το
υπόστρωμα και είχε χημική σύνθεση παρόμοια με αυτήν της οστεΐνης. Η δοκιμασία ΜΤΤ υπέδειξε
μια σταθερή μείωση του πολλαπλασιασμού των κυττάρων επί του στρώματος του ΗΑ σε σύγκριση
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με τα συμβατικά κεραμικά ελέγχου και του σύνθετου υλικού BP67 και για τις τρεις κυτταρικές σειρές
που μελετήθηκαν.
Συμπέρασμα: Η μέθοδος LAB μπορεί να εφαρμοστεί για αύξηση της βιοενεργότητας των υλικών,
τα οποία κανονικά παρουσιάζουν χαμηλή βιοενεργότητα υπό συμβατικές συνθήκες. Αυτό
επιτυγχάνεται με ακριβή έλεγχο των παραμέτρων και των συνθηκών της ακτινοβόλησης με λέιζερ,
ενώ οι κυτταρικές αποκρίσεις χρειάζονται περαιτέρω διερεύνηση.

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