Browse

Pulse frequency dependency of photobiomodulation on osteogenic differentiation of human dental pulp stem cells
펄스변조 광생물조절이 치수줄기세포의 골형성 분화에 미치는 효과 연구

DC Field Value Language
dc.contributor.advisor정종훈-
dc.contributor.author김홍배-
dc.date.accessioned2018-05-28T16:35:24Z-
dc.date.available2018-05-28T16:35:24Z-
dc.date.issued2018-02-
dc.identifier.other000000151211-
dc.identifier.urihttps://hdl.handle.net/10371/140802-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 바이오시스템·소재학부, 2018. 2. 정종훈.-
dc.description.abstractPhotobiomodulation (PBM) therapy contributes to pain relief, wound healing, and tissue regeneration. The pulsed wave (PW) mode has been reported to be more effective than the continuous wave (CW) mode when applying PBM to many biological systems. However, the reason for the higher effectiveness of PW-PBM is poorly understood. Herein, it suggest using delayed luminescence (DL) as a reporter of mitochondrial activity after PBM treatment. DL originates mainly from mitochondrial electron transport chain systems, which produce reactive oxygen species (ROS) and adenosine triphosphate (ATP). The decay time of DL depends on the pulse frequencies of applied light, which correlate with the biological responses of human dental pulp stem cells (hDPSCs). Using a low-power light whose wavelength is 810 nm and energy density is 38 mJ/cm2, it find that a 300-Hz pulse frequency prolonged the DL pattern and enhanced alkaline phosphatase activity. In addition, it analyze mitochondrial morphological changes and their volume density and find evidence supporting mitochondrial physiological changes from PBM treatment. Our data suggest a new methodology for determining the effectiveness of PBM and the specific pulse frequency dependency of PBM in the differentiation of hDPSCs.
In addition, duty cycle and pulse frequency of PW were empirically verified based-on-expanded biology experiment. To do such this, it applied 810 nm LPL of 128 W/cm2 energy density in vitro. Upon this value, CW irradiation did not induce any significant changes for differentiation of hDPSCs. However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the PW with 30 % of duty cycle and 300-3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency.
Meanwhile, together with NIR, blue light has been known to induce differentiation of stem cells. In order to enhance the effectiveness of PBM the blue light is incorporated into NIR-PBM. PBM has almost been used in the region of red or near infrared (NIR) whereas the blue light does not frequently due to inducing disrupt cellular processes. Photoacceptor for NIR is cytochrome c oxidase and is Flavin for blue light in the mitochondrial electron transport chain system. The delayed luminescence (DL), in which radiate light after expose light to cells, originate from cytochrome c oxidase in the region of NIR and from flavins in the region of blue light. Correlation between PBM and NIR and blue light-induced DL for hDPSCs was employed to investigate an optimal excitation to differentiate the cells. The DL showed that the NIR with blue-light-pre-irradiation exerted better excitation than the others (only NIR or blue light) in pulsed wave mode that their energy density was 89 mJ/cm2. In blocking complex I with rotenone, the DL was all decreased in excitation for NIR, blue light and blue-then-NIR light, while Antimycin A-bloocked-complex III DL was preserved for only NIR. It suggests that photoacceptor of blue light may be in complex I and III simultaneously whereas in complex III for NIR. As for differentiation of hDPSCs, it showed that alkaline phosphatase (ALP) activity was highly expressed for blue-then-NIR light. ROS and RT-PCR data support the result of ALP activity.
Based on the results in vitro, PBM was applied to Beagles teeth in order to verify formation of dentin in vivo. A dentin formation was produced to be up to 20 % increment compared to the control that was not exposed to NIR and also induced dense periodontal ligament (PDL) after 8 weeks of exposure of NIR to teeth. It was observed that odontoblast subjected to NIR light exposure was active, generating new tubular dentin along with older dentin. Notably, such active odontoblast was not observed in controls, even though odontoblast-like layer whereby dentin structure was there in SEM analysis. It suggests that such a strategy might facilitate treatments relating to the diseases of dentin and PDL in mature permanent teeth. In summary, PW-PBM was more effective than CW-PWM in differentiating hDPSCs into osteogenesis. Duty cycle 30% and pulse frequency 300 Hz was effective among PW-frequencies. Blue-light-incoporated NIR induced much more differentiation than when only NIR applied. Beagle test verified dentin formation by means of PW-PBM.
-
dc.description.tableofcontentsChapter 1. Photobiomodulation 1
1.1 Light 1
1.1.1 Brief of light 1
1.1.2 Light as photon particles 1
1.1.3 Different light sources 2
1.1.4 Nature of interactions 2
1.1.5 Fate of excited state 5
1.1.6 Light-dental tissue interaction 5
1.2 Photobiomodulation 8
1.2.1 Brief of photobiomodulation 8
1.2.2 Mechanism of PBM 8
1.2.2.1 Strategic mechanism of PBM 8
1.2.2.2 Parameters of PBM 12
1.2.3 Application of PBM to dentistry 15
1.2.3.1 Oral and Maxillofacial indications of PBM 15
1.2.3.2 Human dental pulp-derived stem cells 16
1.2.3.3 Multipotency of human dental pulp-derived stem cell 16
1.2.3.4 Application of PBM to human dental pulp-derived stem cell 16
1.3 Delayed luminescence 19
1.3.1 Delayed luminescence 19
1.3.2 PBM and DL 19
1.4 Measurement of intensity into pulp chamber 20
1.5 Scope of the dissertation 25
Chapter 2. Pulse frequency dependency of photobiomodulation on the bioenergetic functions of human dental pulp stem cells 27
2.1 Introduction 27
2.2 Materials and methods 28
2.2.1 Cell culture 28
2.2.2 PBM system and treatment 30
2.2.3 Delayed luminescence spectroscopy 30
2.2.4 Mathematical description of light-induced DL as a probability distribution 31
2.2.5 Cell viability assay 31
2.2.6 Detection of intracellular ROS 31
2.2.7 Alkaline phosphatase assay 31
2.2.8 Electron transmission microscopy 31
2.2.9 Mitochondrial bioenergetics 32
2.2.10 Statistical analysis 33
2.3 Results 33
2.3.1 PBM-mediated ROS production, proliferation, and alkaline phosphatase activity 33
2.3.2 Delayed luminescence 36
2.3.3 Morphological changes in mitochondria from PBM treatment 38
2.4 Discussion 41
2.5 Conclusion 47
Chapter 3. Effects of pulsing of light on the dentinogenesis of human dental pulp stem cells in vitro 49
3.1 Introduction 49
3.2 Materials and methods 50
3.2.1 Fabrication of PBM system and application to cell culture plates 50
3.2.2 Cell culture 52
3.2.3 Measurement of cytoplasmic membrane potential 54
3.2.4 Alkaline phosphatase (ALP) assay 54
3.2.5 WST- 1 assay 54
3.2.6 Adenine tri-phosphatase (ATP) assay 55
3.2.7 Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP, ψm) 55
3.2.8 Real time polymerase chain reaction 55
3.2.9 Western blotting 56
3.2.10 Statistical analysis 57
3.3 Results and Discussion 57
3.3.1 PBM conditions for effective modulation of hDPSCs activity 57
3.3.2 Intracellular reactive oxygen species (ROS) and mitochondrial responses 62
3.3.3 Nucleus transcription factors 65
3.3.4 Dentinogenic differentiation 66
3.4 Conclusion 74
Chapter 4. Near-infrared irradiation enhance differentiation of human dental pulp stem cells via blue-light-pre-irradiation 75
4.1 Introduction 75
4.2 Materials and methods 75
4.2.1 Cell culture 75
4.2.2 Fabrication of PBM system and Light dose 77
4.2.3 Mathematical description of the light-induced DL as a probability distribution 78
4.2.4 WST-1 measurement 78
4.2.5 Detection of intracellular ROS 79
4.2.6 Alkaline phosphatase (ALP) assay 79
4.2.7 Alizarin Red S staining 82
4.2.8 Real time polymerase chain reaction 82
4.2.9 Electron transmission microscopy 83
4.2.10 Statistical analysis 83
4.3 Results 83
4.3.1 DL analysis 83
4.3.2 Proliferation of hDPSCs 84
4.3.3 Osteogenic differentiation of hDPSCs 84
4.3.4 ROS production and PCR analysis 90
4.4 Discussion 90
4.5 Conclusion 98
Chapter 5. Additional dentin formation in beagles tooth by means of PBM 99
5.1 Introduction 99
5.2 Materials and methods 100
5.2.1 Animal 100
5.2.2 Photobiomodulation 100
5.2.3 Histological procedure 102
5.2.4 Micro-CT tomography 102
5.2.5 Scanning electron microscopy 102
5.2.6 Statistical analysis 103
5.3 Results 103
5.3.1 Development of dentin tissue was confirmed by micro-CT tomography 103
5.3.2 Histological analysis of teeth 103
5.3.3 Structural analysis by SEM 103
5.3.4 Developed periodontal ligament by PBM 105
5.3.5 Vasculature by PBM 105
5.4 Discussion 105
5.5 Conclusion 114
Chapter 6. Summary 115
Reference 117
Abstract in Korean 133
-
dc.formatapplication/pdf-
dc.format.extent6898594 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectPhotobiomodulation-
dc.subjectPulse frequency-
dc.subjectDelayed luminescence-
dc.subjectOsteogenic differentiation-
dc.subjectHuman dental pulp stem cell-
dc.subjectCytochrome c oxidase-
dc.subject.ddc660.6-
dc.titlePulse frequency dependency of photobiomodulation on osteogenic differentiation of human dental pulp stem cells-
dc.title.alternative펄스변조 광생물조절이 치수줄기세포의 골형성 분화에 미치는 효과 연구-
dc.typeThesis-
dc.description.degreeDoctor-
dc.contributor.affiliation농업생명과학대학 바이오시스템·소재학부-
dc.date.awarded2018-02-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Biosystems and Biomaterials Science and Engineering (바이오시스템·소재학부)Theses (Ph.D. / Sc.D._바이오시스템·소재학부)
Files in This Item:
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse