臺灣博碩士論文加值系統

本篇論文探討人類骨母細胞株在利用交流靜電場，給予高電壓低電流的電刺激後產生的反應。以往曾有許多研究探討人類細胞在各種不同外界刺激下的反應，這些外加的刺激多半為直流靜電場，電磁場，直接通電（直流電或交流電），或是利用機械裝置所產生的應力，鮮少有研究觸及交流靜電場。本實驗利用MG-63細胞株作為研究對象，並將其置於1 kV，160 μA的交流靜電場中，利用多種測試方法，包括細胞活性測試，鹼性磷酸酶測試，Von Kossa染色法，亞硝酸鹽測試，反轉錄聚合酵素鏈鎖反應以及同步定量聚合酵素鏈鎖反應來評估交流靜電場對於人類骨母細胞生長之影響。研究的結果顯示在此條件之下，靜電場會抑制骨母細胞表現鹼性磷酸酶的能力，但對於第一型膠原蛋白以及骨鈣素基因之調控則呈現不同的結果。在此參數設定下之靜電場具有抑制第一型膠原蛋白基因活性，以及促進骨鈣素基因活性之能力。此外，細胞存活率和起始細胞密度有關，顯示初始細胞數量在人類骨母細胞生長過程中也是一個重要的因素。

The aim of this study was to determine the effects of an AC electrostatic field generated from a device with high voltage and low current output on human osteoblastic cell line. Many studies about how human cells react and respond to external stimulations of various kinds and types were done before. Most of the external stimulations were DC electrostatic fields, electromagnetic fields, direct electrocution (DC or AC), or even mechanical loadings in these studies, but studies using AC electrostatic field as an external stimulation source were rarely found. This study utilized human osteoblasts as the target cells by putting them under the influence of an AC electrostatic field, which was set to use output parameters of 1 kV and 160 μA. The effects of electrostatic field on human osteoblastic cells were assessed by utilizing various kinds of tests, including cell viability test, alkaline phosphatase assay, Von Kossa staining, nitrite assay, reverse transcriptase PCR and real time quantitative PCR. The results showed that under current condition, the AC electrostatic field had a down-regulation effect on the production ability of alkaline phosphatase. Specific effects on different genes were discovered with the application of EFID. The expression level of osteocalcin gene was elevated while the expression level of type I collagen gene was reduced. In addition, cell viability showed a cell count-dependent result, suggesting initial cell density may also be a crucial factor in the proliferation process of human osteoblasts.

中文摘要..................................................i
ASTRACT..................................................ii
ACKNOWLEDGEMENT.........................................iii
CONTENTS.................................................iv
TABLE CONTENTS..........................................vii
FIGURE CONTENTS........................................viii
CHAPTER 1 INTRODUCTION....................................1
1.1 Fracture healing....................................1
1.2 Osteoblasts.........................................2
CHAPTER 2 LITERATURE REVIEW...............................5
2.1 External stimulations manipulated cell growth.......5
2.1.1 Surface remodeling..............................6
2.1.2 Introduction of chemical compounds..............7
2.1.3 Application of physical stimulations............8
2.2 The history of electrostimulation..................10
2.2.1 Before 20th century............................10
2.2.2 In mid-20th century............................11
2.2.3 The recent years ..............................12
2.3 Electrostimulations and cell development...........12
2.4 Current progress of electrostimulation on
mammalian cell development........................ 13
CHAPTER 3 EFID AND PROBLEM DEFINITION....................19
3.1 The EFID...........................................19
3.2 Problem definition.................................20
3.2.1 Hypothesis and objectives......................21
3.2.2 Testing methods................................22
CHAPTER 4 MATERIALS AND EXPERIMENT SETUP.................23
4.1 Materials..........................................23
4.1.1 Cell culture...................................23
4.1.2 Instruments....................................24
4.1.3 Reagents.......................................24
4.1.4 Expendables....................................30
4.2 Assignation of cells and culture plates............31
4.3 EFID setup.........................................32
CHAPTER 5 INCUBATION OF CELL CULTURES....................34
5.1 Heat inactivation of fetal bovine serum............34
5.2 Complete medium....................................35
5.3 Medium test........................................35
5.4 Thawing cells......................................35
5.5 Passage of adherent cells (subculture).............36
CHAPTER 6 TESTING METHODS................................38
6.1 MTT assay..........................................38
6.2 ALP assay..........................................39
6.3 Von Kossa staining.................................40
6.4 Cell morphology....................................41
6.5 Nitrite assay......................................41
6.6 Gene expression analysis...........................42
6.6.1 RNA extraction.................................42
6.6.2 Determination of RNA concentration.............44
6.6.3 Efficacy check of reverse transcription kit....44
6.6.4 Reverse transcription..........................45
6.6.5 Real-time quantitative PCR.....................46
6.7 Statistical analysis...............................48
CHAPTER 7 RESULTS AND DISCUSSIONS........................50
7.1 Results............................................50
7.1.1 1x10E4 cells/well with and without EFID........50
7.1.2 1x10E5 cells/well with and without EFID........57
7.1.3 Different initial cell density with EFID.......64
7.1.4 Different initial cell density without EFID....71
7.2 Discussions........................................78
7.2.1 1x10E4 cells/well with and without EFID........80
7.2.2 1x10E5 cells/well with and without EFID........81
7.2.3 Different initial cell density with EFID.......83
7.2.4 Different initial cell density without EFID....84
7.3 Limitation and future prospects....................86
CHAPTER 8 CONCLUSIONS....................................88
REFERENCES...............................................89
APPENDIX A Bone remodeling process.......................97
B Osteoclast development ......................100
C Relative quantitation algorithms of
real-time quantitative PCR...................101
D Effects of EFID on mouse macrophages.........104
E Previous experiment data.....................110
F Cell aggregation and condensation............114

[1] A. De Blasio, C. Messina, A. Santulli, V. Mangano, E. Di Leonardo, A. D’Anneo, G. Tesoriere, R. Vento, "Differentiative pathway activated by 3-aminobenzamide, an inhibitor of PARP, in human osteosarcoma MG-63 cells," Federation of European Biochemical Societies Letters, Vol. 579, 2005, pp.615-620.
[2] Alexa O., "Electrically induced osteogenesis. II. Experimental studies," Revista medico-chirurgicală̆ a Societă̆ţ̜ii de Medici ş̧i Naturaliş̧ti din Iaş̧i, Vol. 100, 1996, pp62-65.
[3] Áron Lazáry, Bernadett Balla, Jánós P. Kosa, Krisztián Bácsi, Zsolt Nagi, István Takács, Péter P. Varga, Gábor Speer, Péter Lakatos, "Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells," Biomaterials, Vol. 28, 2007, pp.393-399.
[4] Applied Biosystems, Applied Biosystems Real-Time PCR Systems User Bulletin: Relative Quantitation (RQ) Algorithms in Applied Biosystems Real-Time PCR Systems Software, Foster City: Applied Biosystems, 2007.
[5] Applied Biosystems, Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems User’s Manual, Foster City: Applied Biosystems, 2007.
[6] Applied Biosystems, Application Note Real-Time PCR: Understanding CT, Foster City: Applied Biosystems, 2008.
[7] Ayman Hamed, Paul Kim, and Michael Cho, "Synthesis of nitric oxide in human osteoblasts in response to physiologic stimulation of electrotherapy," Annals of Biomedical Engineering, Vol. 34, no. 12, 2006, pp.1908-1916.
[8] Bellina Veronesi, Guangwei Wei, Jin-Qi Zeng, Marga Oortgiesen, "Electrostatic charge activates inflammatory vanilloid (VR1) receptors," NeuroToxicology, Vol. 24, 2003, pp.463-473.
[9] Benjamin S. Harrison, Anthony Atala, "Carbon nanotube applications for tissue engineering," Biomaterials, Vol. 28, 2007, pp.344-353.
[10] C. A. Bassett, R. J. Pawluk, and A. A. Pilla, "Acceleration of fracture repair by electromagnetic fields: A surgically noninvasive method," Annals of the New York Academy of Sciences, Vol. 238, 1974, pp. 242-262.[11] C. A. Bassett, S. N. Mitchell, and S. R. Gaston, "Treatment of ununited tibial diaphyseal fractures with pulsing electromagnetic fields," The Journal of Bone and Joint Surgery, Vol. 63, Issue 4, 1981, pp. 511-523.
[12] C. H. Lohmann, Z. Schwartz, Y. Liu, Z. Li, B. J. Simon, V. L. Sylvia, D. D. Dean, L. F. Bonewald, H. J. Donahue, B. D. Boyan, "Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells," Journal of Orthopaedic Research, Vol. 21, 2003, pp.326-334.
[13] C. O. Pabo, and R. T. Sauer, "Transcription factors: structural families and principles of DNA recognition," Annual Review of Biochemistry, Vol. 61, 1992, pp. 1053-1095.
[14] C. T. Hung, F. D. Allen, S. R. Pollack, and C. T. Brighton, "What is the role of the convective current density in the real-time calcium response of cultured bone cells to fluid flow?," Journal of Biomechanics, Vol. 29, no. 11, 1996, pp.1403-1409.
[15] Carl T. Brighton, J. Black, Z. B. Friedenberg, J. L. Esterhai, L. J. Day, and J. F. Connolly, "A multicenter study of the treatment of non-union with constant direct current," The Journal of Bone and Joint Surgery, Vol. 63 Issue 1, 1981, pp. 2-13.
[16] Carl T. Brighton and Solomon R. Pollack, "Treatment of recalcitrant non-union with a capacitively coupled electrical field: A preliminary report," The Journal of Bone and Joint Surgery, Vol. 67, Issue 4, 1985, pp. 577-585.
[17] Carl T. Brighton, Wei Wang, Charles C. Clark, "Up-regulation of matrix in bovine articular cartilage explants by electric fields," Biochemical and Biophysical Research Communications, Vol. 342, 2006, pp.556-561.
[18] Carl T. Brighton, Wei Wang, Richard Seldes, Guihong Zhang, and Solomon R. Pollack, "Signal transduction in electrically stimulated bone cells," The Journal of Bone and Joint Surgery, Vol. 83-A, no. 10, 2001, pp.1514-1523.
[19] Carl T. Brighton, Z. B. Friedenberg, L. M. Zemsky, and P. R. Pollis, "Direct-current stimulation of non-union and congenital pseudarthrosis: Exploration of its clinical application," The Journal of Bone and Joint Surgery, Vol. 57, Issue 3, 1975, pp.368-377.
[20] Ching-Hua Hsieh, Molecular Dynamics Simulation to Investigate Diffusion Behavior of Polystyrene in Tetrahydrofuran under External Electric Field, Master''s Thesis, National Sun Yat-Sen University, Kaohsiung, Taiwan, 2001.[21] Christopher J. Edwards and Tim D. Spector, "Statins as modulators of bone formation," Arthritis Research, Vol. 4, 2002, pp.151-153.
[22] Dengshun Miao, Susan Murant, Nanette Scutt, Paul Genever, and Andrew Scutt, "Megakaryocyte—Bone marrow stromal cell aggregates demonstrate increased colony formation and alkaline phosphatase expression in vitro," Tissue Engineering, Vol. 10, no. 5-6, 2004, pp.807-817.
[23] Eleftherios Tsiridis, Neil Upadhyay, Peter Giannoudis, "Molecular aspects of fracture healing: Which are the important molecules?," Injury, Vol. 38, Issue 1, Supplement 1, 2007, pp.S11-S25.
[24] Fitzsimmons R. J., Ryaby T. J., Magee F. P., and Baylink D. J. "Combined magnetic fields increase net calcium flux in bone cells," Calcified Tissue International, Vol. 55, no. 5, 1994, pp.376-380.
[25] Frank L. Tabrah, Philip Ross, Mary Hoffmeier, and Fred Gilbert Jr., "Clinical report on long-term bone density after short-term EMF application," Bioelectromagnetics, Vol. 19, Issue 2, 1998, pp.75-78.
[26] Fukuda E., Yasuda I., "On the piezoelectric effect of bone," Journal of the Physiological Society of Japan, Vol. 12, 1957, pp.1158-1162.
[27] G. Otting, Y. Q. Qian, M. Billeter, M. Müller, M. Affolter, W. J. Gehring, and K. Wüthrich, "Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution," The European Molecular Biology Organization Journal, Vol 9, no 10, 1990, pp. 3085-3092.
[28] G. Scott and J. B. King, "A prospective, double-blind trial of electrical capacitive coupling in the treatment of non-union of long bones," The Journal of Bone and Joint Surgery, Vol 76, Issue 6, 1994, pp. 820-826.
[29] Gehring W. J., Affolter M, Bürglin T., "Homeodomain proteins," Annual Review of Biochemistry, Vol 63, 1994, pp. 487-526.
[30] H. Yoshimoto, Y. M. Shin, H. Terai, J. P. Vacanti, "A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering," Biomaterials, Vol. 24, 2003, pp.2077-2082.
[31] Haugland R. P., K. D. Larison, Handbook of Fluorescent Probes and Research Chemicals 6th ed., Eugene: Molecular Probes, 1996.
[32] Hongming Zhuang, Wei Wang, Richard M. Seldes, A. David Tahernia, HuijunFan, and Carl T. Brighton, "Electrical stimulation induces the level of TGF-β1 mRNA in osteoblastic cells by a mechanism involving calcium/calmodulin pathway," Biochemical and Biophysical Research Communications, Vol. 237, 1997, pp.225-229.
[33] I. Binderman, D. Somjen, Z. Shimshoni, J. Levy, H. Fischler, and R. Kprenstein, "Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific," Biochimica et Biophysica Acta, Vol. 844, 1985, pp.273-279.
[34] Igor Entin, Alex Plotnikov, Rafi Korenstein, and Yona Keisari, "Tumor growth retardation, cure, and induction of antitumor immunity B16 melanoma-bearing mice by low electric field-enhanced chemotherapy," Clinical Cancer Research, Vol. 9, 2003, pp.3190-3197.
[35] I. O. Smith, M. J. Baumann, L. R. McCabe, "Electrostatic interactions as a predictor for osteoblast attachment to biomaterials," Journal of Biomedical Materials Research, Vol. 70A, Issue 3, 2004, pp.436-441.
[36] I. R. Garrett, D. Chen, G. Gutierrez, M. Zhao, A. Escobedo, G. Rossini, S. E. Harris, W. Gallwitz, K. B. Kim, S. Hu, C. M. Crews, and G. R. Mundy, "Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro," The Journal of Clinical Investigation, Vol. 111, 2003, pp.1771-1782.
[37] Jane B. Lian, Gary S. Stein, Amjad Javed, Andre J. van Wijnen, Janet L. Stein, Martin Montecino, Mohammad Q. Hassan, Tripti Gaur, Christopher J. Lengner, and Daniel W. Young, "Networks and hubs for the transcriptional control of osteoblastogenesis," Reviews in Endocrine and Metabolic Disorders, Vol. 7, no. 1-2, 2006, pp.1-16.
[38] Janet Rubin, Kenneth J. McLeod, Louisa Titus, Mark S. Nanes, Bayard D. Catherwood, and Clinton T. Rubin, "Formation of osteoclast-like cells is suppressed by low frequency, low intensity electric fields," Journal of Orthopaedic Research, Vol. 14, Issue 1, 1996, pp.7-15.
[39] Jingdong Deng, Karl H. Schoenbach, E. Stephen Buescher, Pamela S. Hair, Paula M. Fox, and Stephen J. Beebe, "The effects of intense submicrosecond electrical pulses on cells," Biophysical Journal, Vol. 84, 2003, pp.2709-2714.
[40] K. Anselme, "Osteoblast adhesion on biomaterials," Biomaterials, Vol. 21, 2000, pp.667-681.
[41] K. Heermeier, M. Spanner, J. Träger, R. Gradinger, P. G. Strauss, W. Kraus, andJ. Schmidt, "Effects of extremely low frequency electromagnetic field (EMF) on collagen type I mRNA expression and extracellular matrix synthesis of human osteoblastic cells," Bioelectromagnetics, Vol. 19, 1998, pp.222-231.
[42] K. Yonemori, S. Matsunaga, Y. Ishidou, S. Maeda, and H. Yoshida, "Early effects of electrical stimulation on osteogenesis," Bone, Vol. 19, Issue 2, 1996, pp.173-180.
[43] Landry P. S., Sadasivan K. K., Marino A. A., and Albright J. A., "Electromagnetic fields can affect osteogenesis by increasing the rate of differentiation," Clinical Orthopaedics and Related Research , Vol. 338, 1997, pp.262-270.
[44] Leroy S. Lavine, Irving Lustrin, Robert A. Rinaldi, Morris H. Shamos, and Abraham R. Liboff, "Electric enhancement of bone healing," Science, Vol. 175. no. 4026, 1972, pp.1118-1121.
[45] Li Tian, Yu-Xin Huang, Min Tian, Wei Gao, Qing Chang, "Downregulation of electroacupuncture at ST36 on TNF-a in rats with ulcerative colitis," World Journal of Gastroenterology, Vol. 9, no. 5, 2003, pp.1028-1033.
[46] Luca Cucullo, Gabriele Dini, Kerri L. Hallene, Vincent Fazio, Erin V. Ilkanich, Chiazor Igboechi, Kelly M. Kight, Mukesh K. Agarwal, Mary Garrity-Moses, and Damir Janigro, "Very low intensity alternating current decreases cell proliferation," Glia, Vol. 51, 2005, pp.65-72.
[47] M. C. Siebers, K. Matsuzaka, X. F. Walboomers, S. C. G. Leeuwenburgh, J. G. C. Wolke, J. A. Jansen, "Electrostatic spray deposition (ESD) of calcium phosphate coatings, an in vitro study with osteoblast-like cells," Biomaterials, Vol. 25, 2004, pp.2019-2027.
[48] M. C. Siebers, K. Matsuzaka, X. F. Walboomers, S. C. G. Leeuwenburgh, J. G. C. Wolke, J. A. Jansen, "Osteoclastic resorption of calcium phosphate coatings applied with electrostatic spray deposition (ESD) in vitro," Journal of Biomedical Materials Research, Vol. 74A, Issue 4, 2005, pp.570-580.
[49] M. De Mattei, M. Fini, S. Setti, A. Ongaro, D. Gemmati, G. Stabellini, A. Pellati, and A. Caruso, "Proteoglycan synthesis in bovine articular cartilage explants exposed to different low-frequency low-energy pulsed electromagnetic fields," Osteoarthritis and Cartilage, Vol. 15, no. 2, 2007, pp.163-168.
[50] M. Nagai and M. Ota, "Pulsating electromagnetic field stimulates mRNA expression of bone morphogenetic protein-2 and -4," Journal of DentalResearch, Vol. 73, 1994, pp.1601-1605.
[51] Maija P. Valta, Teuvo Hentunen, Qiang Qu, Eeva M. Valve, Anna Harjula, Jani A. Seppänen, H. Kalervo Väänänen, and Pirkko L. Härkönen, "Regulation of osteoblast differentiation: A novel function for fibroblast growth factor 8," Endocrinology, Vol. 147, no. 5, 2006, pp.2171-2182.
[52] Mareke Hartig, Ulrich Joos, Hans-Peter Wiesmann, "Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro," European Biophysics Journal, Vol. 29, 2000, pp.499-506.
[53] Melita M. Dvorak, Ashia Siddiqua, Donald T. Ward, D. Howard Carter, Sarah L. Dallas, Edward F. Nemeth, and Daniela Riccardi, "Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones, "Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, no. 14, 2004, pp.5140-5145.
[54] Michael Cho, Hemant S. Thatte, Raphael C. Lee, and David E. Golan, "Integrin-dependent human macrophage migration induced by oscillatory electrical stimulation," Annals of Biomedical Engineering, Vol. 28, 2000, pp.234-243.
[55] Mohammad Q. Hassan, Amjad Javed, Maria I. Morasso, Jeremy Karlin, Martin Montecino, Andre J. van Wijnen, Gary S. Stein, Janet L. Stein, and Jane B. Lian, "Dlx3 Transcriptional Regulation of Osteoblast Differentiation: Temporal Recruitment of Msx2, Dlx3, and Dlx5 Homeodomain Proteins to Chromatin of the Osteocalcin Gene", Molecular and Cellular Biology, Vol. 24, No. 20, 2004, pp.9248–9261.
[56] Nirupama K. Shevde, Lori A. Plum, Margaret Clagett-Dame, Hironori Yamamoto, J. Wesley Pike, and Hector F. DeLuca, "A potent analog of 1α,25-dihydroxyvitamin D3 selectively induces bone formation, "Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, no. 21, 2002, pp.13487-13491.
[57] Ozawa H., Abe E., Shibasaki Y., Fukuhara T., and Suda T., "Electric fields stimulate DNA synthesis of mouse osteoblast-like cells (MC3T3-E1) by a mechanism involving calcium ions," Journal of Cellular Physiology, Vol. 138, 1989, pp.477-483.
[58] Pamela S. Hair, Karl H. Schoenbach, E. Stephen Buescher, "Sub-microsecond,intense pulsed electric field applications to cells show specificity of effects," Bioelectrochemistry, Vol. 61, 2003, pp.65-72.
[59] R. Korenstein, D. Somjen, H Fischler, and I. Binderman, "Capacitive pulsed electric stimulation of bone cells: Induction of cyclic-AMP changes and DNA synthesis," Biochimica et Biophysica Acta, Vol. 803, 1984, pp.302-307.
[60] Saima N. Nayab, Frances H. Jnes, Irwin Olsen, "Modulation of the human bone cell cycle by calcium ion-implantation of titanium," Biomaterials, Vol. 28, 2007, pp.38-44.
[61] Sang Hyun Kim, Victor J. Johnson, and Raghubir P. Sharma, "Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-κB and p38 MAPK signaling pathways," Nitric Oxide, Vol. 7, 2002, pp.67-74.
[62] Sharrad W. J., "A double-blind trial of pulsed electromagnetic fields for delayed union of tibial fractures," Journal of Bone and Joint Surgery-British Volume, Vol. 72-B, Issue 3, 1990, pp.347-355.
[63] Shmuel Yaccoby, Michele J. Wezeman, Maurizio Zangari, Ronald Walker, Michele Cottler-Fox, Danna Gaddy, Wen Ling, Rinku Saha, Bart Barlogie, Guido Tricot, and Joshua Epstein, "Inhibitory effects of osteoblasts and increased bone formation on myeloma in novel culture systems and a myelomatous mouse model," Haematologica, Vol. 91, no. 2, 2006, pp.192-199.
[64] T. J. Chambers, "The pathobiology of the osteoclast," Journal of Clinical Pathology, Vol. 38, 1985, pp.241-252.
[65] United States Department of Health and Human Services, Public Health Service, Office of the Surgeon General, Bone Health and Osteoporosis: A Report of the Surgeon General, Honolulu, Hawaii: University Press of the Pacific, 2004.
[66] Wei Wang, Shuying Yang, Jianzhong Shao, Yi-Ping Li, "Signaling and transcriptional regulation in osteoblast commitment and differentiation," Frontiers in Bioscience, Vol. 12, 2007, pp.3068-3092.
[67] Wei Wang, Zhenyu Wang, Guihong Zhang, Charles C. Clark, and Carl T. Brighton, "Up-regulation of chondrocyte matrix genes and products by electric fields," Clinical Orthopaedics and Related Research, no. 427S, 2004, pp.S163-S173.
[68] William J. Boyle, W. Scott Simonet, and David L. Lacey, "Osteoclastdifferentiation and activation," Nature, Vol. 423, 2003, pp.337-342
[69] Yasuda I., "Fundamental aspects of fracture treatment," Journal of Kyoto Medical Society, Vol. 4, 1953, pp.395-406.
[70] Z. B. Friedenberg, P. G. Roberts Jr., N. H. Didizian, and Carl T. Brighton, "Stimulation of fracture healing by direct current in the rabbit fibula," The Journal of Bone and Joint Surgery, Vol.53, 1971, pp.1400-1408.
[71] Zhenyu Wang, Charles C. Clark, and Carl T. Brighton, "Up-regulation of bone morphogenetic proteins in cultured murine bone cells with use of specific electric fields," The Journal of Bone and Joint Surgery, Vol. 88-A, no. 5, 2006, pp.1053-1065.
[72] Zhong-Liang Deng, Katie A. Sharff, Ni Tang, Wen-Xin Song, Jinyong Luo, Xiaoji Luo, Jin Chen, Erwin Bennett, Russell Reid, David Manning, Antia Xue, Anthony G. Montag, Hue H. Luu, Rex C. Haydon, and Tong-Chuan He, "Regulation of osteogenic differentiation during skeletal development," Frontiers in Bioscience, Vol. 13, 2008, pp.2001-2021.
[73] 詹惠雯，探討脈衝式電磁場促進類骨母細胞增生之機轉，碩士論文，私立中原大學醫學工程學系，桃園，2003。
[74] 陳俐婷，脈衝列電磁場刺激對骨母細胞生物活性的影響，碩士論文，私立中原大學醫學工程學系，桃園，2003。
[75] 李冠瑢，電磁場與超音波刺激對骨母細胞生長影響之比較，博士論文，私立中原大學醫學工程學系，桃園，2003。
[76] 鄭益利，超音波對人類軟骨細胞的生物效應，碩士論文，國立中山大學生物科學研究所，高雄，2005。
[77] 何宜靜，超高分子量聚乙烯磨耗顆粒對巨噬細胞生長及細胞激素分泌的影響，碩士論文，國立臺北科技大學生物科技研究所，台北，2006。