||Low Power Laser Irradiation on the Proliferation, Osteo-Differentiation, and Anti-Inflammation of Stem Cells
||Department of BioMedical Engineering
Low power laser irradiation
Bone marrow stem cells
Adipose-derived stem cells
Rat calvarial bone defect model
本研究結果顯示，低能量雷射照射不會對幹細胞產生毒性。同時，低能量雷射在能量密度4 J/cm2時會顯著的促進幹細胞增生。在骨分化方面，隨著雷射照射能量的增加有促進骨分化的能力。同時，低能量雷射也會促進骨分化相關基因的表現並減少蝕骨作用相關因子的表現。藉由IGF1與BMP2的抗體所進行的實驗也顯示，低能量雷射可能藉由調節IGF1或BMP2訊息傳遞路徑去調節幹細胞的生長和分化。此外，根據動物實驗的結果可知在大鼠頭蓋骨缺損處之PLGA支架無論是否有幹細胞的置放，LPLI照射皆可增加骨頭癒合的程度。而幹細胞與LPLI共同處理的組別則具有最佳的治癒效果。另一方面，幹細胞會表現Toll-like receptors，並且藉由LPS的刺激會顯著地引起發炎相關基因(Cox-2, Il-1β, IL-6, and IL-8)的表現，¬若同時處理能量密度8 J/cm2的低能量雷射，會對發炎相關基因的表現有最佳的抑制效果。本研究也發現，低能量雷射會降低磷酸化IκBα以及磷酸化NF-κB的表現，同時減少轉錄因子NF-κB轉位的現象以及轉錄的活性。此外，LPLI透過提升細胞內之cAMP濃度而抑制NF-κB之轉錄活性，經由SQ22536 (cAMP抑制物)處理過後，LPLI所引起之抗發炎作用將被消除，因此可知LPLI之抗發炎效果是透過cAMP之調控所造成。
Low power laser irradiation (LPLI) has been wildly applied in treating a variety of clinical diseases, such as wound healing, pain relief, and anti-inflammatory reaction. In addition, stem cells have the properties of self-renewal and multi-differentiation, which make the stem cells possess the potential application in repair the damage or diseased tissues and organs. Osteoporosis is a common skeletal disorder to induce frequent bone fracture. Based on the ability of osteogenic differentiation of stem cells, it may provide a new approach of the treatment for osteoporosis and osteoporosis facture. The microbial infection is a common complication during the stem cell based therapy or transplantation which restricts the applicability of stem cell therapy. The purposes of this study are to investigate the effect of low power laser on the proliferation of stem cells, then, study the mechanism of LPLI stimulates the osteogenic differentiation of stem cells from the in vitro and in vivo experiments. Finally, the effect of LPLI on the LPS-induced inflammation of stem cells was investigated.
The results in this study found that no cytotoxic effects were observed on irradiated stem cells. LPLI significantly promoted proliferation of stem cells at 4 J/cm2 and enhanced osteogenic differentiation in a dose-dependent manner. Expression of the osteogenic markers was significantly increased by LPLI. Contrarily, LPLI decreased the expression of osteoclastogenic markers (RANKL/OPG). The antibodies neutralization experiments indicated that physiological effects of LPLI may regulate IGF1 and BMP2 signaling pathways to control cell proliferation and/or osteogenic differentiation. Based on the rat calvarial bone defect experiment, LPLI displayed higher amounts of newly generated bone on both stem cell loaded groups and non-stem cells loaded groups. LPLI plus stem cells group showed the best healing outcome in this study. hADSCs expressed the TLR1, 2, 3, 4, and 6, and significantly induced the production of pro-inflammatory mediators (Cox-2, Il-1β, IL-6, and IL-8). LPLI remarkably inhibited these gene expressions with the optimal dose of 8 J/cm2, and decreased the protein level of phosphor-IκBα and phospho-NF-κB. The amount of nuclear translocation and transcriptional activity of NF-κB was decreased by LPLI. The inhibitory effect stimulated by LPLI might act via increasing the intracellular level of cAMP, resulting in down-regulation of NF-κB transcriptional activity.
The present study suggests that LPLI promotes the proliferation and osteogenic differentiation of stem cells. LPLI also suppresses the inflammatory response of LPS-induced inflammation of stem cells. These results may provide insight for further investigations of the application of LPLI to stem cells in regenerative medicine and the potential for anti-inflammatory therapy followed by stem cell therapy. In addition, one hypothesis has been mentioned that LPLI can act as a photo-mechanical stimulation on the cells. Therefore, the mechanical properties of stem cells followed by LPLI will be discussed in the future study.
Table of contents VII
List of tables X
List of figures XI
Glossary of Acronyms XVII
Chapter 1: Introduction 1
1.1 Exordium 1
1.2 Introduction of lasers 2
1.2.1 The development and applications of low power laser irradiation 2
1.2.2 The physiological role and possible mechanism induced by LPLI 4
1.3 stem cells 6
1.3.1 Source of stem cells 6
1.3.2 Bone marrow mesenchymal stem cells 8
1.3.3 Adipose derived stem cells 8
1.4 Bone tissue 10
1.4.1 The function and structure of bone 10
1.4.2 Bone remodeling cycle 11
1.4.3 Osteoporosis 13
1.4.4 Bone healing process 14
1.5 Immunology 16
1.5.1 Innate immune response 16
1.5.2 Toll-like receptors 17
1.5.3 TLRs-mediated MyD88-independent signaling pathway 19
1.6 Purposes 21
1.6.1 LPLI on the proliferation and osteogenic differentiation of mBMSCs 21
1.6.2 LPLI on the inhibitory effect of LPS-induced inflammation of hADSCs 21
Chapter 2: Materials and methods 23
2.1 Flowchart 23
2.2 Laser apparatus 24
2.3 Cell culture 26
2.3.1 D1 mouse bone marrow stem cells 26
2.3.2 Human adipose derived stem cells 27
2.3.3U937 human leukemic monocyte lymphoma cell 27
2.3.4 Osteogenic differentiation 28
2.4 Animals 28
2.5 Cell cytotoxicity 28
2.5.1 Cell morphology 28
2.5.2 Lactate dehydrogenase (LDH) assay 29
2.6 Cell viability 30
2.6.1 Cells counting 30
2.6.2 MTT assay 30
2.6.3 MTS assay 31
2.7 Functional assay of osteogenic differentiation 31
2.7.1 Alkaline phosphatase (ALP) activity assay 31
2.7.2 Alizarin Red S (ARS) stain 33
2.8 Real-time reverse transcription-polymerase chain reaction (RT-PCR) 34
2.8.1 Total RNA extraction 34
2.8.2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 35
2.9 Enzyme-linked immunosorbent assay (ELISA) 38
2.9.1 ELISA for IGF1 38
2.9.2 ELISA for BMP2 39
2.9.3 ELISA for IL-6 and IL-8 39
2.9.4 ELISA for Cyclic AMP 40
2.10 Neutralization 41
2.11 Fabrication of PLGA scaffolds 41
2.12 Cell culture on PLGA scaffolds 42
2.13 Animal procedures (critical-sized calvarial defect) 43
2.14 Micro-CT analysis 44
2.15 LPS treatment 45
2.16 Western blotting 45
2.16.1 Solution preparation 45
2.16.2 Protein sample preparation and concentration measurement 46
2.16.3 Gel electrophoresis 46
2.16.4 Electrotransfer 48
2.16.5 Immunoblotting 48
2.17 NFκB activity assay 49
2.17.1 Plasmid DNA purification 49
2.17.2 Dual-luciferases reporter gene assay 50
2.18 Immunofluorescence 51
2.19 Statistical analysis 52
Chapter 3: Results 53
3.1 The cytotoxicity and viability of LPLI on stem cells 53
3.1.1 Cell cytotoxicity 53
3.1.2 Effect of LPLI on cell viability and the Laser parameters 55
3.2 LPLI on the osteogenic differentiation of mBMSCs 60
3.2.1 LPLI enhances osteogenic differentiation by functional assay 60
3.2.2 LPLI regulates osteogenic genes expressions 63
3.2.3 The biophysiological effects of LPLI 65
3.3 Effect of LPLI on the in vivo bone regeneration 70
3.3.1 hADSCs remained in PLGA scaffold 70
3.3.2 LPLI modestly promotes bone repair by micro-CT analysis 71
3.4 The anti-inflammatory effect of LPLI on hADSCs 76
3.4.1 Determination the TLRs expression pattern and activity in hADSCs 76
3.4.2 LPLI decreased LPS-induced pro-inflammatory gene expression 78
3.4.3 LPLI downregulates LPS-stimulated NF-κB activation 83
3.4.4 LPLI modulated NF-κB via the cAMP signaling pathway 87
Chapter 4: Discussion and conclusion 90
4.1 LPLI on the proliferation and osteogenic differentiation of mBMSCs 90
4.2 LPLI on the bone repair in rat calvarial defect model 94
4.3 LPLI on the inhibitory effect of LPS-induced inflammation of hADSCs 97
Chapter 5: Future work 101
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