臺灣博碩士論文加值系統

組織工程是一項結合細胞、支架、生物反應器與訊息因子之跨領域。本論文主要是結合具生物降解性之孔洞支架、三維式生物反應器與脈衝式電磁場刺激，調控不同階段之骨生成細胞（造骨細胞與骨髓間葉幹細胞）在體外之三維式與二維式骨生成。本論文主要分為兩大主軸：（I）結合脈衝式電磁場與生物反應器並發展為一新型體外培養整合系統，以調控大鼠造骨細胞在三維式培養環境中之生長；以及（II）利用脈衝式電磁場刺激調控人類骨髓間葉幹細胞在二維環境下之骨分化表現。
在實驗（I）中，首先利用初生大鼠頭蓋骨分離出造骨前趨細胞，並以5105的細胞密度植於細胞支架中。細胞支架之製備主要為利用聚乳酸-甘醇酸聚合物與溶劑融合鹽析法製造多孔性支架。將種植於支架之細胞培養於三維式生物反應器，每天接受2小時與8小時的脈衝式電磁場刺激，電磁場參數為：具300 s波寬的單脈衝波形、頻率7.5 Hz、磁通量密度分別為0.13、0.24與0.32 mT。在實驗（II）中，將人類骨髓間葉幹細胞分別培養於生長型與骨分化型培養液中，並利用實驗（I）中已找出的最佳電磁場刺激參數（磁通量密度0.13 mT，以及每天刺激2小時），以探討長期與短期之脈衝式電磁場調控間葉幹細胞之增生與骨分化的能力。
實驗（I）之結果顯示，短時間低強度之電磁場刺激（2 hr、0.13 mT）於培養期間可顯著促進大鼠造骨細胞之增生；中強度（0.24 mT）刺激在培養末期抑制細胞數目；而高強度電磁場刺激（0.32 mT）則不論刺激時間長短，皆抑制細胞之增生。相反地，高強度電磁場刺激（0.32 mT）則明顯促進造骨細胞之整體鹼性磷酸酵素活性。經由H&E染色則證實在2小時的電磁場刺激下（0.13 mT），造骨細胞在支架中具有最佳的細胞增生表現。實驗（II）之結果則顯示，在骨分化培養環境與長期電磁場刺激下，電磁場刺激可於培養第七天時提升間葉幹細胞分泌鹼性磷酸酵素之能力，並在第十天時促進細胞之增生，在低初始密度之組別尤其明顯。此外，骨生成之早期基因，如：Runx2/Cbfa1與鹼性磷酸酵素，同樣受到脈衝式電磁場之調控而在第七日有最高之表現量；組織染色結果則顯示間葉幹細胞之骨分化與骨礦化在培養最後一天達到最高表現量。而間葉幹細胞在短期電磁場刺激後之細胞數目與鹼性磷酸酵素之活性則無明顯變化。
因此，本論文證實具特定參數之脈衝式電磁場刺激不但可於體外調控造骨細胞與間葉幹細胞之骨生成作用，此新型體外培養整合系統更具有應用於硬骨組織工程之潛能。因此，本篇研究顯示脈衝式電磁場對於未來在再生醫學之應用不啻為一有效的利器。

Tissue engineering is an interdisciplinary field integrated with cells, scaffolds, bioreactors, and signals. The intent of this thesis is to modulate the osteogenesis of two osteogenic cells (osteoblasts and bone marrow-derived mesenchymal stem cells) in 3-D and 2-D culture by integrating biodegradable porous scaffolds, bioreactors, and pulsed electromagnetic field (PEMF). The thesis is divided into two major parts: (I) to develop a novel system integrated with PEMF and bioreactors to modulate murine osteoblast growth in 3-D culture, and (II) to identify the modulatory role of PEMF on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in 2-D culture.
In Experiment I, murine osteoblasts acquired from neonatal calvaria were seeded onto porous poly (DL-lactic-co-glycolic acid) scaffolds fabricated by the solvent merging/ particulate leaching method. Cell matrices cultured in bioreactors were exposed to daily PEMF stimulation with an induced electric waveform consisting of single, narrow 300 us quasi-rectangular pulses with a repetition rate of 7.5 Hz, and with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT. In Experiment II, hMSCs isolated from adult bone marrow were cultured with basal and osteogenic medium for up to 28 days and exposed to daily 2-hr PEMF stimulation with average amplitude of 0.13 mT for 2 hr. The modulatory effects of PEMG with long-term and short-term exposure on hMSC osteogenesis were characterized.
In Experiment I, the cell number of murine osteoblasts was significantly enhanced by 2-hr PEMF stimulation at 0.13 mT. PEMF stimulation with higher amplitudes (i.e. 0.24 and 0.32 mT) showed significant inhibitory effect on cell proliferation, especially at 0.32 mT (irrespective of exposure time). However, PEMF stimulation at 0.32 mT significantly enhanced the total alkaline phosphatase (ALP) activity produced by osteoblasts. In addition, osteoblasts exposed to 2-hr PEMF stimulation at 0.13 mT expressed better cell adhesion and growth onto scaffolds by H&E staining. In Experiment II, hMSCs at lower initial cell density cultured in osteogenic culture expressed relatively higher ALP activity at day 7, followed by greater cell numbers at day 10 under long-term PEMF exposure. Furthermore, the highest expression of early osteogenic genes, including Runx2/Cbfa1 and ALP, was also observed at day 7 by PEMF exposure. Based on the histochemical staining, the osteogenic differentiation and matrix mineralization reached the highest levels at day 28. However, hMSCs exposed to short-term PEMF pre-treatment showed similar cell number and ALP activity compared to their controls during the whole culture period.
In conclusion, it indicated that not only PEMF stimulation with specific parameters had a biological effect on modulating the proliferation and osteogenic differentiation of murine osteoblasts and hMSCs in vitro, but this novel integration cultivation system also had great potential on bone tissue engineering applications. Taken together, it provides insights on the development of PEMF as an effective technology for regenerative medicine.

Table of Content
中文摘要 I
Abstract II
Acknowledgement III
Table of Content V
List of Figures VI
List of Tables VIII
Chapter 1 Scope of Thesis 1
Scope of Thesis 3
Chapter 2 Introduction 5
2-1 Bone Tissue Engineering 7
2-2 Stem Cells 26
2-3 Pulsed Electromagnetic Fields (PEMF) 36
2-4 References 44
Chapter 3 Pulsed Electromagnetic Fields Affect Osteoblast Proliferation and Differentiation in Bone Tissue Engineering 53
3-1 Introduction 55
3-2 Materials and Methods 58
3-3 Results 72
3-4 Discussion 82
3-5 Conclusion 86
3-6 References 87
Chapter 4 Modulation of Osteogenesis in Human Mesenchymal Stem Cells by Specific Pulsed Electromagnetic Field Stimulation 91
4-1 Introduction 92
4-2 Materials And Methods 94
4-3 Results 100
4-4 Discussion 110
4-5 Conclusion 112
4-6 References 113
Chapter 5 Conclusions and Perspectives 117
Conclusion and Perspectives 119
Appendix Journal Paper ― A A–1
Appendix Journal Paper ― B B–1
Appendix Biographical Sketch C–1

List of Figures
Figure 1.The essential components of bone tissue engineering. 8
Figure 2.Bone cell populations. 10
Figure 3.The histological structure of trabecular bone. 12
Figure 4.The localization of bone cells. 13
Figure 5.3D scaffold systems of various porosity and pore geometry fabricated by fused deposition modeling 19
Figure 6.Different kinds of spinner flasks designed for three-dimensional cell/tissue culture in vitro. 20
Figure 7.The rotating wall vessel bioreactors. 22
Figure 8.The flow perfusion bioreactor. 23
Figure 9.Origin of stem cells in the mammalian embryo. 28
Figure 11.Hematopoietic and stromal stem cell differentiation, and its plasticity. 32
Figure 12.Osteoblastic differentiation is regulated by some of the dominant signals. 33
Figure 13.Intramembranous ossification and endochondral ossification. 35
Figure 14.Different lateral x-ray films of tibiae taken from a female patient.. 38
Figure 15.Transverse microradiographs of the middle of the diaphyseal shaft of the ulna of the turkey after eight weeks if treatment.. 40
Figure 16.Time course for PEMF-stimulated [3H] thymidine incorporation two types of cells cultured in the presence of different serum concentrations. 43
Figure 17.Schematic of the integration system. 57
Figure 18.Murine osteoblast primary culture. 59
Figure 19.Diagram of PLGA scaffold fabrication. 61
Figure 20.Prototypes of bioreactors. 62
Figure 21.Novel bioreactor system at Generation III combined with PEMF stimulation system. 63
Figure 22.PEMF signals. 64
Figure 23.Different induced electric intensities used in this study. 65
Figure 24.Cell seeding onto PLGA scaffolds. 67
Figure 25.Selected signals of magnetic flux densities (i.e., 0.13 mT, 0.24 mT, and 0.32 mT) applied to this study. 68
Figure 26.Schematic diagram of experimental process. 69
Figure 27.Integration system of PEMF stimulator and bioreactors. 69
Figure 28.PLGA scaffolds. 72
Figure 29.Light micrographs of H&E-stained osteoblasts seeded onto PLGA scaffolds. 73
Figure 30.Numbers of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.13 mT) at 0, 6, 12, and 18 days. 75
Figure 31.Numbers of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.24 mT) at 0, 6, 12, and 18 days. 76
Figure 32.Numbers of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.32 mT) at 0, 6, 12, and 18 days. 77
Figure 33.ALP activity of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.13 mT) at 0, 6, 12, and 18 days. 79
Figure 34.ALP activity of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.24 mT) at 0, 6, 12, and 18 days. 80
Figure 35.ALP activity of osteoblasts cultured in PLGA scaffolds with/without PEMF stimulation (0.32 mT) at 0, 6, 12, and 18 days. 81
Figure 36.Diagram of isolation, expansion, and culture of hMSCs. 93
Figure 37.Schematic of PEMF stimulation system 95
Figure 38.Experimental design. 96
Figure 39.Flow chart of total RNA isolation using Qiagen RNeasy Micro Kit. 99
Figure 40.Proliferation and ALP activity of hMSCs at different initial cell seeding densities (1,500 cells/cm2 and 3,000 cells/cm2) during 14-day osteogenic culture. 101
Figure 41.Early osteoblastic gene expression of Runx2/Cbfa1, ALP, and collagen type I during hMSC osteogenesis. 103
Figure 42.Osteogenic differentiation and matrix mineralization of hMSCs under PEMF exposure during 28-day osteogenic culture. 104
Figure 43.DNA quantitation of hMSCs cultured in basal medium and osteogenic medium for the first 7 days and the subsequent 14 days during the culture period. 106
Figure 44.ALP activity of hMSCs cultured in basal medium and osteogenic medium for the first 7 days and the subsequent 14 days during the culture period. 107
Figure 45.Osteogenic differentiation and matrix mineralization of hMSCs exposed to short-term PEMF pre-treatment in basal and osteogenic culture. 109

List of Tables
Table 1. Select critical considerations in bone tissue-engineering scaffold design 15
Table 2.Definition of terms for biomaterials designing as scaffolds for bone tissue engineering 16
Table 3.Functional properties of natural and engineered tissues 17
Table 4.Comparison of the different sources of stem cells 27
Table 5.The experimental design of cells/scaffolds in each experiment. 66
Table 6.Gene-specific primers for RT-PCR. 99

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