臺灣博碩士論文加值系統

椎間盤退化是造成背痛的主要原因之一。為了暸解椎間盤退化的病理因素及調控機制。我們建立了使用椎間盤穿刺造成大鼠尾巴椎間盤退化的動物模式，並於穿刺後第四天及第一、二、六、十二週的時間點，探討組織學上及第一二型膠原蛋白表現上與對照組的差異。蛋白質表現上第一型膠原蛋白在穿刺後遞增顯示椎間盤往纖維化型態表現。第二型膠原蛋白則短暫上升後遞減。在組織學上亦表現出椎間盤穿刺後相較於對照組有較快的退化表現。在其它因子表現上，急性期第一介白質(IL-1)及缺氧誘導因子-2 (HIF-2)上升伴隨第十三基質金屬蛋白酶(MMP13)上升。同時我們發現對照組中的大鼠椎間盤，其缺氧誘導因子-2(HIF-2)會隨著老化過程逐漸上升。我們認爲大鼠尾巴穿刺模式可導致椎間盤提早退化並有助於瞭解椎間盤退化的成因。
為了探討中醫藥在治療椎間盤退化的可能性，我們選擇了具有抑制缺氧誘導因子及第十三基質金屬蛋白酶的厚朴萃取物厚朴酚(honokiol)，來進行中醫藥在椎間盤的藥物動力學實驗。因為椎間盤是人體最大的無血管分佈器官，藥物是否可以進入椎間盤具有其治療意義。我們使用高效液相層析儀來進行體外及及大鼠體內的藥物濃度分析。在體外實驗部分，發現厚朴酚可以藉由濃度差擴散進入椎間盤。在動物體內實驗部分，厚朴酚以每公斤30毫克劑量藉由靜脈注射進入體內，分別於注射後15、30、60、120和240分鐘採集血液及椎間盤進行濃度分析。實驗結果發現厚朴酚可以藉由被動擴散進入椎間盤。在有血液供給的情況下，有助於厚朴酚更快分佈於椎間盤。大鼠以每公斤30毫克靜脈注射後，椎間盤及血漿濃度比在15分鐘約36%，在注射30分鐘後濃度比可達到49.6%。實驗結果顯示中醫藥在治療椎間盤退化具有其可行性。至於厚朴酚對於治療椎間盤的退化是否真的有幫助，還需要進一步的實驗證實。

English Abstract
Intervertebral disc (IVD) degeneration is believed to contribute to low back pain. To understand the pathogenesis and regulatory mechanism of puncture-induced IVD degeneration, we established a rat-tail puncture model using Kirschner wire and a homemade stopper. The progress of disc degeneration was evaluated by histological examination and the quantitative measurement of type I and type II collagen expression at 0.5, 1, 2, 6, and 12 weeks after puncture and compared to control rats of the same age. The punctured disc demonstrated transient upregulation of Col2a1 and continuous upregulation of Col1a1 in nucleus pulposus. Histological examination and Safranin-O staining revealed progressive degeneration of the punctured disc. Matrix metalloproteinase 13 (MMP13) was increased at 1 week after puncture but did not change in the control group. Interleukin-1 beta (IL-1) mRNA expression level was elevated at the acute stage after puncture compared to the control group. A transient increase and then decrease in expression of hypoxia inducible factor 2 (HIF-2) in the nucleus pulposus (NP) was detected, followed by an increase at 12 weeks after puncture, while HIF-2 was upregulated with age in control nucleus pulposus. This model may potentially be valuable for the study of the molecular mechanisms and dissection of the pathways underlying disc degeneration.
Honokiol is one of the major compounds of Magnolia officinalis with ability to inhabit hypoxia inducible factor pathway and MMP13 expression. Honokiol can be a possible candidate in the treatment of intervertebral disc degeneration. To evaluate the distribution of honokiol into intervertebral disc, we developed in vitro and in vivo methods using high-performance liquid chromatography. The rat tail disc was used as the in vitro experimental model. For in vivo animal experiment, the blood samples and tail discs were collected from cardiac puncture and rat tail separately at 15, 30, 60, 120 and 240 min after honokiol administration (30 mg/kg, i.v.). A validated liquid chromatographic system was applied for honokiol analysis. The in vitro experimental results demonstrated that honokiol diffused into intervertebral disc and was concentration-dependent. Honokiol also distributed easily into the intervertebral disc after honokiol administration (30 mg/kg, i.v.). The concentration ratio of honokiol disc-to-plasma (Cdisc/Cplasma) is about 36% (1.79/4.91) at 15 min and about 49.6% (1.51/3.04) at 30 min after honokiol administration. These results demonstrated that application of traditional Chinese medicine in the treatment of intervertebral disc degeneration should be feasible. However, the therapeutic effect of honokiol in retardation of intervertebral disc degeneration should be further studied.

TABLE OF CONTENTS
Table of Contents …………………………………………….…………..i
Acknowledgements ……………………………………………...………v
中文摘要 ………………………………………………………...vi
English Abstract …………………………………………………….. viii

1. Introduction
1.1 Function of spine ………………………..………………………….1
1.2 The structure and growth of intervertebral disc ……………………1
1.3 Specific characteristics of intervertebral disc ……………………….3
1.4 Degeneration of intervertebral dis …………………………………..4
1.5 The role of cytokines and matrix-degrading enzymes in
intervertebral disc degeneration ……………………………………5
1.6 The role of hypoxia inducible factors in intervertebral disc
degeneration ………………………………………………………...6
1.7 Animal models in the study of intervertebral degeneration ………..8
1.8 The potentials of traditional Chinese medicine in intervertebral
degeneration ………………………………………………………..9
1.9 Brief introduction of honokiol …………………………..………...10
1.9.1 Pharmacokinetics of Honokiol ………………………………...10
1.9.2 Therapeutic effects of Honokiol ……………………………….11
2. Study motives and aims of study …………………………………….12
3. Materials and Methods
3.1 Establishment and time course investigation of intervertebral disc
degeneration in a rat-tail puncture model ………………………... 14
3.1.1 Rationale ……………………………………………………...14
3.1.2 Experimental approach …………………………………...…..14
3.1.2.1 Annulus puncturing of rat tail discs …………………….....14
3.1.2.2 Histological staining ……………………………………....15
3.1.2.3 Immunohistochemistry staining …………………………..16
3.1.2.4 Quantitative PCR (qPCR) …………………………………17
3.1.2.5 ELISA for HIF-2 ………………………………………...17
3.1.2.6 Western Blotting analysis …………………………………18
3.1.2.7 Statistical analyses ………………………………………...19
3.2 In vitro penetration and in vivo distribution of honokiol into
intervertebral disc in rat …………………………………………...19
3.2.1 Rationale ……………………………………………………...19
3.2.2 Experimental approach ……………………..………………20
3.2.2.1 Chemical and reagents …………………………………….20
3.2.2.2 HPLC for Analysis of Honokiol in rat plasma and
intervertebral disc …………………………………………20
3.2.2.3 To measure the honokiol concentration into the intervertebral
disc in vitro ………………………………………………22
3.2.2.4 To measure the Honokiol concentration into the
intervertebral disc in vivo …………………………………23
4. Results
4.1 Establishment and time course investigation of intervertebral disc
degeneration in a rat-tail puncture model …………………………25
4.1.1 Transient upregulation of Col2a1 and continuous upregulation
of Col1a1 in nucleus pulposus after puncturing of the
intervertebral disc ……………………………………………25
4.1.2 Histological changes after puncturing of the intervertebral
disc …………………………………………………………...25
4.1.3 HIF-2 and IL-1 are overexpressed during the acute stage after
disc puncture …………………………………………………26
4.1.4 MMP13 is upregulated after disc puncture …………………..27
4.2 In vitro penetration and in vivo distribution of honokiol into
intervertebral disc in rat …………………………………………...28
4.2.1 Validation of a reversed-phase HPLC method for analysis of
honokiol in rat plasma and intervertebral disc ………………28
4.2.2 Passive diffusion of honokiol into intervertebral disc in in vitro
study …………………………………………………………30
4.2.3 Honokiol concentration is reachable within therapeutic level in
in vivo study ...………………………………………………..30
4.2.4 The passive diffusion property of honokiol is governed by the
blood supply of intervertebral disc …………………………..31
5. Discussion
5.1 Establishment of intervertebral disc degeneration model using
rat-tail puncture …………………………………………………...33
5.2 IL-1β induction by puncture could increase MMP13 expression and
promote degeneration of the intervertebral disc …………………..34
5.3 HIF-2 expression increased in acute stage and marked increased
at 12 weeks after puncture ………………………………………...36
5.4 Delivery ability of substances should be evaluated in intervertebral
disc ………………………………………………………………...36
5.5 The penetration of honokiol into intervertebral disc is reliable to
achieve therapeutic concentration after honokiol administration …37
5.6 The pharmacokinetics and possible side effects of honokiol ……..38
6. Conclusion …………………………………………………………...41
7. Reference …………………………………………………………….43
8. Figure legend .……………………….……………………………….58
9. Table list ……………………………………………………………..84
10. Appendices …………………………………………………………90

1. Andersson, G. B. Epidemiological features of chronic low-back pain. The Lancet 1999, 354, 581–585.
2. Dagenais, S.; Caro, J.; Haldeman, S. A systematic review of low back pain cost of illness studies in the United States and internationally. The Spine Journal 2008, 8, 8–20.
3. Grange, L.; Gaudin, P.; Trocme, C. Intervertebral disc degeneration and herniation: the role of metalloproteinases and cytokines. Revue du … 2001.
4. Adams, M. A.; Roughley, P. J. What is intervertebral disc degeneration, and what causes it? Spine 2006, 31, 2151–2161.
5. Pattappa, G.; Li, Z.; Peroglio, M.; Wismer, N.; Alini, M.; Grad, S. Diversity of intervertebral disc cells: phenotype and function. J Anat 2012, no–no.
6. Choi, K.; Cohn, M.; Harfe, B. Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation. Dev Dyn 2008, 237, 3953–3958.
7. Risbud, M. V.; Schaer, T. P.; Shapiro, I. M. Toward an understanding of the role of notochordal cells in the adult intervertebral disc: from discord to accord. Dev Dyn 2010, 239, 2141–2148.
8. Lewis, K. E.; Eisen, J. S. Hedgehog signaling is required for primary motoneuron induction in zebrafish. Development 2001, 128, 3485–3495.
9. Christ, B.; Wilting, J. From somites to vertebral column. Annals of Anatomy - Anatomischer Anzeiger 1992, 174, 23–32.
10. Horner, H. A.; Urban, J. P. 2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine 2001, 26, 2543–2549.
11. Huang, Y.-C.; Urban, J. P. G.; Luk, K. D. K. Intervertebral disc regeneration: do nutrients lead the way? Nature Publishing Group 2014, 10, 561–566.
12. Urban, J.; Smith, S.; Fairbank, J. Nutrition of the intervertebral disc. Spine 2004, 29, 2700–2709.
13. Raj, P. P. Intervertebral disc: anatomy-physiology-pathophysiology-treatment. Pain Pract 2008, 8, 18–44.
14. Wilke, H. J.; Neef, P.; Caimi, M.; Hoogland, T.; Claes, L. E. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine 1999, 24, 755–762.
15. Hsieh, A. H.; Twomey, J. D. Cellular mechanobiology of the intervertebral disc: new directions and approaches. J Biomech 2010, 43, 137–145.
16. Geiss, A.; Larsson, K.; Rydevik, B.; Takahashi, I.; Olmarker, K. Autoimmune properties of nucleus pulposus: an experimental study in pigs. Spine 2007, 32, 168–173.
17. Geiss, A.; Larsson, K.; Junevik, K.; Rydevik, B.; Olmarker, K. Autologous nucleus pulposus primes T cells to develop into interleukin-4-producing effector cells: an experimental study on the autoimmune properties of nucleus pulposus. J Orthop Res 2009, 27, 97–103.
18. Kepler, C. K. C.; Anderson, D. G. D.; Tannoury, C. C.; Ponnappan, R. K. R. Intervertebral disk degeneration and emerging biologic treatments. J Am Acad Orthop Surg 2011, 19, 543–553.
19. Freemont, A. J. The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 2009, 48, 5–10.
20. Le Maitre, C. L.; Pockert, A.; Buttle, D. J.; Freemont, A. J.; Hoyland, J. A. Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans 2007, 35, 652–655.
21. Hunter, C.; Matyas, J.; Duncan, N. Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. J Anat 2004, 205, 357–362.
22. Peacock, A. Observations on the pre-natal development of the intervertebral disc in man. J Anat 1951, 85, 260.
23. Yang, F.; Leung, V. Y. L.; Luk, K. D. K.; Chan, D.; Cheung, K. M. C. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol 2009, 218, 113–121.
24. Izon, M. Collagen fibrillogenesis in the development of the annulus fibrosus of the intervertebral disc. European Cells and Materials 2010, 22, 226–241.
25. Longo, U. G.; Petrillo, S.; Franceschetti, E.; Maffulli, N.; Denaro, V. Growth Factors and Anticatabolic Substances for Prevention and Management of Intervertebral Disc Degeneration. Stem Cells International 2012, 2012, 1–9.
26. Pfirrmann, C.; Metzdorf, A.; Zanetti, M.; Hodler, J. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine 2001.
27. Iatridis, J. C.; Setton, L. A.; Weidenbaum, M.; Mow, V. C. Alterations in the mechanical behavior of the human lumbar nucleus pulposus with degeneration and aging. J Orthop Res 1997, 15, 318–322.
28. Phillips, K. L. E.; Jordan-Mahy, N.; Nicklin, M. J. H.; Le Maitre, C. L. Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Annals of the rheumatic diseases 2013, 72, 1860–1867.
29. Le Maitre, C. L.; Freemont, A. J.; Hoyland, J. A. Human disc degeneration is associated with increased MMP 7 expression. Biotech Histochem 2006, 81, 125–131.
30. Rutges, J.; Kummer, J.; Oner, F.; Verbout, A.; Castelein, R.; Roestenburg, H.; Dhert, W.; Creemers, L. Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels. J Pathol 2008, 214, 523–530.
31. Masuda, K.; Oegema, T. R.; An, H. S. Growth factors and treatment of intervertebral disc degeneration. Spine 2004, 29, 2757–2769.
32. Turgut, M.; Oktem, G.; Uslu, S.; Yurtseven, M. E.; Aktuğ, H.; Uysal, A. The effect of exogenous melatonin administration on trabecular width, ligament thickness and TGF-beta(1) expression in degenerated intervertebral disk tissue in the rat. J Clin Neurosci 2006, 13, 357–363.
33. Hegewald, A. A.; Cluzel, J.; Krüger, J. P.; Endres, M.; Kaps, C.; Thomé, C. Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells. Journal of Orthopaedic Surgery and Research 2014, 9, 73.
34. Taylor, J. Growth of human intervertebral discs and vertebral bodies. J Anat 1975, 120, 49.
35. Li, H.; Liang, C. Z.; Chen, Q.-X. Regulatory Role of Hypoxia Inducible Factor in the Biological Behavior of Nucleus Pulposus Cells. Yonsei Med J 2013, 54, 807.
36. Cavadas, M. A.; Nguyen, L. K.; Cheong, A. Hypoxia-inducible factor (HIF) network: insights from mathematical models. Cell Communication and Signaling 2013, 11, 1–1.
37. Schipani, E. Hypoxic regulation of nucleus pulposus cell survival: from niche to notch. American Journal of Pathology 2010, 176, 1577.
38. Papandreou, I.; Cairns, R. A.; Fontana, L.; Lim, A. L.; Denko, N. C. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2005, 3, 187–197.
39. Richardson, S. M.; Knowles, R.; Tyler, J.; Mobasheri, A.; Hoyland, J. A. Expression of glucose transporters GLUT-1, GLUT-3, GLUT-9 and HIF-1alpha in normal and degenerate human intervertebral disc. Histochem Cell Biol 2008, 129, 503–511.
40. Agrawal, A.; Guttapalli, A.; Narayan, S.; Albert, T. J.; Shapiro, I. M.; Risbud, M. V. Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am. J. Physiol., Cell Physiol. 2007, 293, C621–31.
41. Risbud, M. V.; Guttapalli, A.; Stokes, D. G.; Hawkins, D.; Danielson, K. G.; Schaer, T. P.; Albert, T. J.; Shapiro, I. M. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J. Cell. Biochem. 2006, 98, 152–159.
42. Scortegagna, M.; Ding, K.; Oktay, Y.; Gaur, A.; Thurmond, F.; Yan, L.-J.; Marck, B. T.; Matsumoto, A. M.; Shelton, J. M.; Richardson, J. A.; Bennett, M. J.; Garcia, J. A. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Genet 2003, 35, 331–340.
43. Oktay, Y.; Dioum, E.; Matsuzaki, S.; Ding, K.; Yan, L. J.; Haller, R. G.; Szweda, L. I.; Garcia, J. A. Hypoxia-inducible Factor 2  Regulates Expression of the Mitochondrial Aconitase Chaperone Protein Frataxin. Journal of Biological Chemistry 2007, 282, 11750–11756.
44. Aprelikova, O.; Wood, M.; Tackett, S.; Chandramouli, G. V. R.; Barrett, J. C. Role of ETS transcription factors in the hypoxia-inducible factor-2 target gene selection. Cancer Research 2006, 66, 5641–5647.
45. Yang, S.; Kim, J.; Ryu, J.-H.; Oh, H.; Chun, C.-H.; Kim, B. J.; Min, B. H.; Chun, J.-S. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat Med 2010, 16, 687–693.
46. Patel, S. A.; Simon, M. C. Biology of hypoxia-inducible factor-2alpha in development and disease. Cell Death & Differentiation 2008, 15, 628–634.
47. Masuda, K.; Aota, Y.; Muehleman, C.; Imai, Y.; Okuma, M.; Thonar, E. J.; Andersson, G. B.; An, H. S. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine 2005, 30, 5–14.
48. Larson, J. W.; Levicoff, E. A.; Gilbertson, L. G.; Kang, J. D. Biologic modification of animal models of intervertebral disc degeneration. J Bone Joint Surg Am 2006, 88 Suppl 2, 83–87.
49. Han, B.; Zhu, K.; Li, F.-C.; Xiao, Y.-X.; Feng, J.; Shi, Z.-L.; Lin, M.; Wang, J.; Chen, Q.-X. A simple disc degeneration model induced by percutaneous needle puncture in the rat tail. Spine 2008, 33, 1925–1934.
50. Lotz, J. C. Animal models of intervertebral disc degeneration: lessons learned. Spine 2004, 29, 2742–2750.
51. Chen, F.-P.; Chen, T.-J.; Kung, Y.-Y.; Chen, Y.-C.; Chou, L.-F.; Chen, F.-J.; Hwang, S.-J. Use frequency of traditional Chinese medicine in Taiwan. BMC Health Serv Res 2007, 7, 26.
52. Henrotin, Y.; Clutterbuck, A. L.; Allaway, D.; Lodwig, E. M.; Harris, P.; Mathy Hartert, M.; Shakibaei, M.; Mobasheri, A. ReviewBiological actions of curcumin on articular chondrocytes. Osteoarthr Cartil 2010, 18, 141–149.
53. Vitetta, L.; Cicuttini, F.; Sali, A. Alternative therapies for musculoskeletal conditions. Best Pract Res Clin Rheumatol 2008, 22, 499–522.
54. Chen, Y. J.; Tsai, K. S.; Chan, D. C.; Lan, K. C.; Chen, C. F.; Yang, R.-S.; Liu, S.-H. Honokiol, a low molecular weight natural product, prevents inflammatory response and cartilage matrix degradation in human osteoarthritis chondrocytes. J Orthop Res 2013, 32, 573–580.
55. Vavilala, D. T.; Ponnaluri, V. K. C.; Vadlapatla, R. K.; Pal, D.; Mitra, A. K.; Mukherji, M. Honokiol inhibits HIF pathway and hypoxia-induced expression of histone lysine demethylases. Biochem Biophys Res Commun 2012, 422, 369–374.
56. Tsai, T. H.; Chou, C. J.; Cheng, F. C.; Chen, C. F. Pharmacokinetics of honokiol after intravenous administration in rats assessed using high-performance liquid chromatography. J. Chromatogr. B, Biomed. Appl. 1994, 655, 41–45.
57. Wu, X.; Chen, X.; Hu, Z. High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma. Talanta 2003, 59, 115–121.
58. Böhmdorfer, M.; Maier-Salamon, A.; Taferner, B.; Reznicek, G.; Thalhammer, T.; Hering, S.; Hüfner, A.; Schühly, W.; Jäger, W. In vitro metabolism and disposition of honokiol in rat and human livers. J. Pharm. Sci. 2011, 100, 3506–3516.
59. Jun-Jun, W.; Xiao-Lei, M.; Jing-Ya, C.; Yong, C. The Pharmacokinetics and Tissue Distribution of Honokiol and its Metabolites in Rats. Eur J Drug Metab Pharmacokinet 2016, 41, 587–594.
60. Kim, B. H.; Cho, J. Y. Anti-inflammatory effect of honokiol is mediated by PI3K/Akt pathway suppression. Nature Publishing Group 2008, 29, 113–122.
61. Chen, F.; Wang, T.; Wu, Y.-F.; Gu, Y.; Xu, X.-L.; Zheng, S.; Hu, X. Honokiol: a potent chemotherapy candidate for human colorectal carcinoma. World J Gastroenterol 2004, 10, 3459–3463.
62. Bai, X. Honokiol, a Small Molecular Weight Natural Product, Inhibits Angiogenesis in Vitro and Tumor Growth in Vivo. Journal of Biological Chemistry 2003, 278, 35501–35507.
63. Shen, J.-L.; Man, K.-M.; Huang, P.-H.; Chen, W.-C.; Chen, D.-C.; Cheng, Y.-W.; Liu, P.-L.; Chou, M.-C.; Chen, Y.-H. Honokiol and Magnolol as Multifunctional Antioxidative Molecules for Dermatologic Disorders. Molecules 2010, 15, 6452–6465.
64. Woodbury, A. Neuro-modulating effects of honokiol: a review. 2013, 1–6.
65. PhD, N. L. M.; MD, C. W.; PhD, Z. X. M.; PhD, M. H. M.; PhD, S.-Y. Y. M. Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain. The Spine Journal 2016, 16, 1231–1237.
66. Yu, P.-F.; Jiang, H.; Liu, J.-T.; Li, X.-C.; Qian, X.; Han, S.; Ma, Z.-J. Traditional Chinese Medicine Treatment for Ruptured Lumbar Disc Herniation: Clinical Observations in 102 Cases. Orthop Surg 2014, 6, 229–235.
67. Chen, C.-H.; Chiang, C.-J.; Wu, L.-C.; Yang, C.-H.; Kuo, Y.-J.; Tsuang, Y.-H.; Tsai, T.-H. Time Course Investigation of Intervertebral Disc Degeneration in a Rat-tail Puncture Model. Life Sciences 2016.
68. Chen, C.-H.; Chiang, C.-J.; Wu, L.-C.; Yang, C.-H.; Kuo, Y.-J.; Tsai, T.-H. In vitro Penetration and in vivo Distribution of Honokiol into the Intervertebral Disc in Rat. Anal Sci 2015, 31, 1297–1302.
69. Lin, C.-C.; Wu, Y.-T.; Yen, J.-C.; Chiang, C.-J.; Tsuang, Y.-H.; Tsai, T.-H. In vitro and in vivo methods to measure the ceftriaxone distribution into the rat tail intervertebral disc. Anal Sci 2010, 26, 979–982.
70. Urban, J. P. G.; Smith, S.; Fairbank, J. C. T. Nutrition of the intervertebral disc. Spine 2004, 29, 2700–2709.
71. Bashir, A.; Gray, M. L.; Hartke, J.; Burstein, D. Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI. Magn Reson Med 1999, 41, 857–865.
72. Rde, W. T.; Batten, J. J.; Want, S. A new in-vitro model to investigate antibiotic penetration of the intervertebral disc. Journal of Bone & … 1995.
73. Riley, L. H.; Banovac, K.; Martinez, O. V.; Eismont, F. J. Tissue distribution of antibiotics in the intervertebral disc. Spine 1994, 19, 2619–2625.
74. Alini, M.; Eisenstein, S. M.; Ito, K.; Little, C.; Kettler, A. A.; Masuda, K.; Melrose, J.; Ralphs, J.; Stokes, I.; Wilke, H.-J. Are animal models useful for studying human disc disorders/degeneration? Eur Spine J 2008, 17, 2–19.
75. Antoniou, J.; Steffen, T.; Nelson, F.; Winterbottom, N.; Hollander, A. P.; Poole, R. A.; Aebi, M.; Alini, M. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 1996, 98, 996–1003.
76. Roberts, S.; Caterson, B.; Menage, J.; Evans, E. H.; Jaffray, D. C.; Eisenstein, S. M. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 2000, 25, 3005–3013.
77. Zhang, H.; La Marca, F.; Hollister, S. J.; Goldstein, S. A.; Lin, C.-Y. Developing consistently reproducible intervertebral disc degeneration at rat caudal spine by using needle puncture. Journal of Neurosurgery: Spine 2009, 10, 522–530.
78. Zhang, H.; Yang, S.; Wang, L.; Park, P.; La Marca, F.; Hollister, S. J.; Lin, C.-Y. Time course investigation of intervertebral disc degeneration produced by needle-stab injury of the rat caudal spine: laboratory investigation. Journal of Neurosurgery: Spine 2011, 15, 404–413.
79. Le Maitre, C. L. C.; Freemont, A. J. A.; Hoyland, J. A. J. Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol 2004, 204, 47–54.
80. Maidhof, R.; Jacobsen, T.; Papatheodorou, A.; Chahine, N. O. Inflammation Induces Irreversible Biophysical Changes in Isolated Nucleus Pulposus Cells. PLoS ONE 2014, 9, e99621.
81. Gorth, D. J.; Mauck, R. L.; Chiaro, J. A.; Mohanraj, B.; Hebela, N. M.; Dodge, G. R.; Elliott, D. M.; Smith, L. J. IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro. Arthritis Res Ther 2012, 14, R179.
82. Le Maitre, C. L.; Freemont, A. J.; Hoyland, J. A. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther 2004, 7, R732–R745.
83. Husa, M.; Liu-Bryan, R.; Terkeltaub, R. Shifting HIFs in osteoarthritis. Nat Med 2010, 16, 641–644.
84. DAHESHIA, M.; YAO, J. The Interleukin 1β Pathway in the Pathogenesis of Osteoarthritis. The Journal of Rheumatology 2008, 35, 2306.
85. Huang, B.; Zhuang, Y.; Li, C.-Q.; Liu, L.-T.; Zhou, Y. Regeneration of the Intervertebral Disc With Nucleus Pulposus Cell-Seeded Collagen II/Hyaluronan/Chondroitin-6-Sulfate Tri-Copolymer Constructs in a Rabbit Disc Degeneration Model. Spine 2011, 36, 2252–2259.
86. Berkowitz, A. L.; Miller, M. B.; Mir, S. A.; Cagney, D.; Chavakula, V.; Guleria, I.; Aizer, A.; Ligon, K. L.; Chi, J. H. Glioproliferative Lesion of the Spinal Cord as a Complication of “Stem-Cell Tourism.” New England Journal of Medicine 2016, 375, 196–198.
87. Purmessur, D.; Schek, R. M.; Abbott, R. D.; Ballif, B. A.; Godburn, K. E.; Iatridis, J. C. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells. Arthritis Res Ther 2011, 13, R81.
88. Yang, C.-H.; Chiang, Y.-F.; Chen, C.-H.; Wu, L.-C.; Liao, C.-J.; Chiang, C.-J. The effect of annular repair on the failure strength of the porcine lumbar disc after needle puncture and punch injury. Eur Spine J 2016, 25, 906–912.
89. Roberts, S.; Menage, J.; Urban, J. P. Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine 1989, 14, 166–174.
90. Urban, J. P.; Holm, S.; Maroudas, A.; Nachemson, A. Nutrition of the intervertebral disc: effect of fluid flow on solute transport. Clin Orthop Relat Res 1982, 296–302.
91. Jackson, A. R.; Yuan, T.-Y.; Huang, C.-Y.; Brown, M. D.; Gu, W. Y. Nutrient Transport in Human Annulus Fibrosus is Affected by Compressive Strain and Anisotropy. Ann Biomed Eng 2012, 40, 2551–2558.
92. Day, A. J.; Bao, Y.; Morgan, M.; Williamson, G. Conjugation position of quercetin glucuronides and effect on biological activity. Free Radical Biology and … 2000, 29, 1234–1243.
93. Zhang, Q.; Li, J.; Zhang, W.; An, Q.; Wen, J.; Wang, A.; Jin, H.; Chen, S. Acute and sub-chronic toxicity studies of honokiol microemulsion. REGULATORY TOXICOLOGY AND PHARMACOLOGY 2015, 71, 428–436.