臺灣博碩士論文加值系統

BACKGROUND: Titanium dental implants with non-treated surfaces or treated surfaces have been used for many years. However, it has been discovered that titanium has several drawbacks, such as grey and non-esthetic color, metallic allergy, and increased tendency to plaque adhesion when exposed to the oral cavity. Therefore, a similar material with comparable strength to titanium but improving drawbacks of titanium material is being researched and developed by many scientists worldwide. Usage of zirconia material as a metal replacement is widely popular lately in the field of prosthetic dentistry and begin trying to use as an implant abutment and pontic crown and obtain successful results. One downside of zirconia material is its bioinert smooth surface, leading to poor cell-material interaction. So, using a GDP to modify its surface with allylamine and attached biologic fibronectin proteins to improve its surface properties. Creating a biomimetic environment can increase the reaction with the extracellular matrix proteins and cells attachment, which will lead to improve the cell material interaction and stimulate the osteogenesis. Therefore, in this paper, we analyzed the biological and physical properties of the zirconia surface to study whether it could be a better option to use as a dental implant material with improved surface properties.

OBJECTIVE OF THE STUDY: To obtain a zirconia dental implant material with improved biological and physical properties. Enhanced surface properties may promote the adhesion, proliferation and differentiation of osteoblast-like cells on the zirconia implant material and stimulate the osteogenesis.
MATERIALS AND METHODS: Zirconia disk surfaces treated with GDP-Argon first with three different watts (50W,75W and 85W): “Ar-GDP 50W, Ar-GDP 75W and Ar-GDP 85W” and followed by GDP-Allylamine: “GDP-AA 50W, GDP-AA 75W and GDP-AA 85W”. Then, they were immersed in fibronectin solution with 2 different concentrations (5µg/ml and 10µg/ml) to get our final samples: “AA50W+Fib 5, AA50W+Fib 10 and AA75W+Fib 5, AA75W+Fib 10 and AA85W+Fib 5, AA85W+Fib10”. They have undergone the analysis of physic to check the surface properties and biologic to analyze the viability, differentiation and osteogenic properties after the surface modification.
RESULTS: From surface topography evaluation, irregular folding proteins were in fibronectin grafted disks, and a rough irregular raised granular pattern was observed for allylamine grafted samples. According to EDS and XPS data, carbon and nitrogen elements were increased with increasing allylamine wattage for both allylamine and fibronectin grafted samples while oxygen component was decreased. Average surface roughness (Ra) becomes higher with higher fibronectin concentration (Ra value- Fib10µg/ml > Fib5µg/ml) and allylamine energy. For surface wettability, the contact angle becomes higher with increasing allylamine concentration. However, fibronectin grafted samples show higher hydrophilicity with lower contact angle and higher surface energy than their allylamine counterparts and control sample.
Cell viability analysis data obtained on day5 showed that AA75W+Fib5, AA85W+Fib10, AA50W+Fib5, and AA50W+Fib10 had higher OD values compared to other samples groups and were statistically significant with other groups. ALP activity assay demonstrated that AA85W+Fib10, AA50W+Fib10, and AA85W+Fib5 groups had higher ALP activity in Day5. AA50W+Fib10 group had the highest upregulation of OC, DLX5 and SP7 gene while AA85W+Fib10 group showed highest expression for OPG gene.
CONCLUSION: From the above findings of this study, it can be concluded that fibronectin grafted disks showed significant improvement in both physical and biological analysis. It overcome the genuine bio-inert properties and enhanced the osteoblast-like cell response of adhesion, proliferation and differentiation which is a key aspect of bone formation and remodeling. In addition, osteogenic related gene expression also higher after fibronectin grafting and the best and optimal conditions to be used were AA85W+Fib10 and AA50W+Fib 10. However, further in vivo studies should be conducted to confirm the result obtained in this in vitro study and the efficacy of fibronectin protein coating on the implant surface.

BACKGROUND: Titanium dental implants with non-treated surfaces or treated surfaces have been used for many years. However, it has been discovered that titanium has several drawbacks, such as grey and non-esthetic color, metallic allergy, and increased tendency to plaque adhesion when exposed to the oral cavity. Therefore, a similar material with comparable strength to titanium but improving drawbacks of titanium material is being researched and developed by many scientists worldwide. Usage of zirconia material as a metal replacement is widely popular lately in the field of prosthetic dentistry and begin trying to use as an implant abutment and pontic crown and obtain successful results. One downside of zirconia material is its bioinert smooth surface, leading to poor cell-material interaction. So, using a GDP to modify its surface with allylamine and attached biologic fibronectin proteins to improve its surface properties. Creating a biomimetic environment can increase the reaction with the extracellular matrix proteins and cells attachment, which will lead to improve the cell material interaction and stimulate the osteogenesis. Therefore, in this paper, we analyzed the biological and physical properties of the zirconia surface to study whether it could be a better option to use as a dental implant material with improved surface properties.

OBJECTIVE OF THE STUDY: To obtain a zirconia dental implant material with improved biological and physical properties. Enhanced surface properties may promote the adhesion, proliferation and differentiation of osteoblast-like cells on the zirconia implant material and stimulate the osteogenesis.
MATERIALS AND METHODS: Zirconia disk surfaces treated with GDP-Argon first with three different watts (50W,75W and 85W): “Ar-GDP 50W, Ar-GDP 75W and Ar-GDP 85W” and followed by GDP-Allylamine: “GDP-AA 50W, GDP-AA 75W and GDP-AA 85W”. Then, they were immersed in fibronectin solution with 2 different concentrations (5µg/ml and 10µg/ml) to get our final samples: “AA50W+Fib 5, AA50W+Fib 10 and AA75W+Fib 5, AA75W+Fib 10 and AA85W+Fib 5, AA85W+Fib10”. They have undergone the analysis of physic to check the surface properties and biologic to analyze the viability, differentiation and osteogenic properties after the surface modification.
RESULTS: From surface topography evaluation, irregular folding proteins were in fibronectin grafted disks, and a rough irregular raised granular pattern was observed for allylamine grafted samples. According to EDS and XPS data, carbon and nitrogen elements were increased with increasing allylamine wattage for both allylamine and fibronectin grafted samples while oxygen component was decreased. Average surface roughness (Ra) becomes higher with higher fibronectin concentration (Ra value- Fib10µg/ml > Fib5µg/ml) and allylamine energy. For surface wettability, the contact angle becomes higher with increasing allylamine concentration. However, fibronectin grafted samples show higher hydrophilicity with lower contact angle and higher surface energy than their allylamine counterparts and control sample.
Cell viability analysis data obtained on day5 showed that AA75W+Fib5, AA85W+Fib10, AA50W+Fib5, and AA50W+Fib10 had higher OD values compared to other samples groups and were statistically significant with other groups. ALP activity assay demonstrated that AA85W+Fib10, AA50W+Fib10, and AA85W+Fib5 groups had higher ALP activity in Day5. AA50W+Fib10 group had the highest upregulation of OC, DLX5 and SP7 gene while AA85W+Fib10 group showed highest expression for OPG gene.
CONCLUSION: From the above findings of this study, it can be concluded that fibronectin grafted disks showed significant improvement in both physical and biological analysis. It overcome the genuine bio-inert properties and enhanced the osteoblast-like cell response of adhesion, proliferation and differentiation which is a key aspect of bone formation and remodeling. In addition, osteogenic related gene expression also higher after fibronectin grafting and the best and optimal conditions to be used were AA85W+Fib10 and AA50W+Fib 10. However, further in vivo studies should be conducted to confirm the result obtained in this in vitro study and the efficacy of fibronectin protein coating on the implant surface.

Table of Contents
Table of content -i-
Figure list -iii-
Table list -v-
Abbreviation list -vi-
ABSTRACT - 1 -
CHAPTER I. INTRODUCTION - 4 -
1.1 General background - 4 -
1.2 Objective of the study - 8 -
1.2 Hypothesis of the study - 8 -
CHAPTER II. LITERATURE REVIEW - 9 -
2.1 Zirconia dental implant - 9 -
2.2 Surface modification techniques - 11 -
2.3 Glow discharge plasma (GDP) - 12 -
2.4 Fibronectin - 15 -
2.5 GDP and fibronectin grafting - 16 -
CHAPTER III. MATERIAL AND METHODS - 19 -
3.1 Zirconia disks(Zr disks) - 19 -
3.2 Zirconia disks preparation - 19 -
3.2.1 Glow discharge plasma (GDP) cleaning - 19 -
3.2.2 Protein grafting - 19 -
3.2.3 Cell culturing and seeding on the zirconia surface - 20 -
3.2.3 Sample size and statistical analysis - 22 -
3.3 Zirconia surface physical analysis - 22 -
3.3.1 Scanning electron microscopic observation (SEM) - 22 -
3.3.2 Concentration analysis of fibronectin - 23 -
3.3.3 Elemental analysis by energy-dispersive X-ray spectroscopy (EDS) - 23 -
3.3.4 X-ray photoelectron spectroscopy analysis (XPS) - 24 -
3.3.5 Functional group analysis by Attenuated Total Reflection Fourier transformed infrared spectroscopy (ATR-FTIR) - 25 -
3.3.6 Surface wettability analysis - 25 -
3.3.7 Surface roughness analysis by Optical profiler using White light interferometer (WLI) - 26 -
3.4 Zirconia surface biological analysis - 27 -
3.4.1 Cell viability analysis and confocal laser scanning microscopy (CLSM) - 27 -
3.4.2 Alkaline Phosphatase Activity Assay (ALP) - 28 -
3.4.3 Reverse Transcriptase qualitative Polymerase Chain Reaction (RT-qPCR) - 28 -
CHAPTER IV. RESULTS - 31 -
4.1 Zirconia surface physical analysis - 31 -
4.1.1 Scanning electron microscopic observation (SEM) - 31 -
4.1.2 Concentration analysis by FITC labelling - 31 -
4.1.3 Elemental analysis by energy-dispersive X-ray spectroscopy (EDS) - 32 -
4.1.4 X-ray photoelectron spectroscopy analysis (XPS) - 33 -
4.1.5 Functional group analysis by Attenuated Total Reflection Fourier transformed infrared spectroscopy (ATR-FTIR) - 34 -
4.1.6 Surface wettability analysis - 34 -
4.1.7 Surface roughness analysis by Optical profiler using White light interferometer (WLI) - 36 -
4.2 Zirconia surface biological analysis - 37 -
4.2.1 Cell viability analysis and CLSM - 37 -
4.2.2 ALP activity assay - 38 -
4.2.3 Reverse Transcriptase qualitative Polymerase Chain Reaction (RT-qPCR) - 39 -
CHAPTER V. DISCUSSION - 41 -
5.1 Glow discharge plasma (GDP) treatment and fibronectin grafting - 41 -
5.2 Zirconia surface physical analysis - 43 -
5.2.1 Surface morphology by using SEM and concentration analysis - 43 -
5.2.2 Elemental analysis by energy-dispersive X-ray spectroscopy (EDS) - 44 -
5.2.3 X-ray photoelectron spectroscopy analysis (XPS) - 45 -
5.2.4 Surface wettability analysis - 45 -
5.2.5 Surface roughness analysis by Optical profiler using White light interferometer (WLI) - 47 -
5.3 Zirconia surface biological analysis - 49 -
5.3.1 Cell viability and CLSM analysis - 49 -
5.3.2 ALP assay activity - 52 -
5.3.3 Reverse Transcriptase qualitative Polymerase Chain Reaction (RT-qPCR) - 53 -
CHAPTER VI. CONCLUSION - 56 -
REFERENCE - 57 -
APPENDIX - 88 -




Figure list

Figure 1. Photo of Glow discharge plasma and interior view of glow discharge plasma processing
Figure 2. Glow discharge plasma machine
Figure 3. Flow chart of processing the zirconia disk
Figure 4. Description of the role of fibronectin in Osseointegration
Figure 5. List of total groups after processing the fibronectin grafting
Figure 6. Photo of total groups of all samples I
Figure 7. Photo of total groups of all samples II
Figure 8. Scanning electron microscope(SU-3500; Hitachi Ltd., Kyoto, Japan)
Figure 9. Energy Dispersive Spectroscope(SU-3500; Hitachi Ltd., Kyoto, Japan)
Figure 10. X-ray Photoelectron Spectroscope(ESCALAB, VG Scientific, East Grinstead, UK)
Figure 11. Attenuated Total Reflection Fourier transformed infrared spectroscope(ATR-FTIR) from Nicolet iS5 (Thermo Fisher Scientific, Madison, WI, USA)
Figure 12. Optical Profiler (TR200, An-Bomb instrument CO., Ltd., Tainan, Taiwan)
Figure 13. Gbx Digidrop Contact Angle Meter(Dublin, Ireland)
Figure 14. Stellaris 8 Confocal laser microscope (Leica, Germany)
Figure 15. SpectraMax iD3 Multi-Mode Microplate Reader from Molecular Devices, USA
Figure 16. Equipment for qPCR (a)Nanodrop;, (b)Thermal Cycler; and (c)LightCycler 96 Instrument; Roche, Switzerland electron microscope
Figure 17. Scanning electron microscope images showing the samples of (a)Control, (b)AA50W, (c)AA50W+Fib5, (d)AA50W+Fib10, (e)AA75W, (f)AA75W+Fib5, (g)AA75W+Fib10, (h)AA85W, (i)AA85W+Fib5 and (j)AA85W+Fib10 in magnification of 60x, 200x, 1000x and 2000x
Figure 18. Fibronectin concentration analysis by FITC labelling (a) AA50W+Fib5, (b) AA50W+Fib10, (c) AA75W+Fib5, (d) AA75W+Fib10, (e) AA85W+Fib5, (f) AA85W+Fib10
Figure 19. showing the FTIR result of (a)50Watt group-AA50W, AA50W+Fib5, AA50W+Fib10, (b)75Watt group-AA75W, AA75W+Fib5, AA75W+Fib10, (c)85Watt group-AA85W, AA85W+Fib5, AA85W+Fib10 groups
Figure 20. Surface wettability evaluation of (a) all groups, (b) 50W group, (c) 75W group and (d) 85W group
Figure 21. Surface roughness evaluation of (a) all groups, (b) 50W group, (c)75W group and (d)85W group
Figure 22. Correlation chart between contact angel and concentration of allylamine and fibronectin
Figure 23. Correlation chart between surface roughness and concentration of allylamine and fibronectin
Figure 24. Cell viability evaluation of (a)all groups, (b)50W group, (c)75W group and (d)85W group
Figure 25. ALP activity assay of (a)all groups, (b)50W group, (c)75W group and (d)85W group
Figure 26. Gene expression of OC
Figure 27. Gene expression of DLX5
Figure 28. Gene expression of SP7
Figure 29. Gene expression of OPG
Figure 30 and 31. Scatter plot showing the mean value of MTT and ALP on Day1, Day3 and Day5
Figure 32. Immunofluorescent assay pictures by confocal laser scanning microscope

 
Table list

Table 1. showing the Energy dispersive spectroscopy Data of AA50W, AA50W+Fib5, AA50W+Fib10, AA75W, AA75W+Fib5, AA75W+Fib10, AA85W, AA85W+Fib5 and AA85W+Fib10
Table 2. showing the result of X-ray Photoelectron Spectroscopy of Control, AA50W, AA50W+Fib5, AA50W+Fib10, AA75W, AA75W+Fib5, AA75W+Fib10, AA85W, AA85W+Fib5 and AA85W+Fib10 groups
Table 3. Primer complete names
Table 4. Primer sequences for real-time polymerase chain reaction
 

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