臺灣博碩士論文加值系統

隨著醫療進步，我們知道類固醇廣泛應用於疾病上治療，如內分泌疾病、風濕性疾病、皮膚疾病、過敏疾病等，而類固醇的使用也是造成非創傷性股骨頭壞死的危險因素之一。儘管類固醇在臨床治療上有極大貢獻，卻使得患者可能有罹患股骨頭壞死的風險，不過目前類固醇誘導骨壞死的機制尚未明確。在台灣此疾病常發生於45-65歲中壯年人口(1)，而這個族群為家庭主要的經濟來源。股骨頭壞死初期無症狀易被忽略，如果不及時治療，有超過 70 % 股骨頭壞死的患者將導致髖關節破壞與股骨頭崩塌，最後終將走向人工髖關節置換的命運，由於股骨頭壞死的發生會造成生活上不便利性，故在治療崩塌前的預防即為重要的課題(2)。本研究目的為使用股骨頭壞死動物模型建立探討單一脈衝式電磁場、福善美 (alendronate) 應用於類固醇誘發股骨頭壞死骨質改變之效應，進行新療法開發，期望可就由此研究找到股骨頭崩塌前的治療。

材料方法
本實驗使用 70 隻 12 週大白鼠隨機分派 7 組，分為對照組、負控制組、正控制組、單一脈衝式電磁場組 (包括高頻率單一脈衝式電磁場與低頻率單一脈衝式電磁場) 及合併治療組 (包括高頻率單一脈衝式電磁場合併 alendronate 與低頻率單一脈衝式電磁場合併 alendronate。每兩天注射糖皮質類固醇 (40 mg/kg methylprednisolone) 及 4 週負重訓練誘導股骨頭壞死隻動物模型，之後分別應用單一脈衝式電磁場及單一脈衝式電磁場合併 alendronate 治療兩種方式做 6 週治療，分別命名為單一脈衝式電磁場組及合併治療組。

結果
首先由外觀觀察，在各組皆未有崩塌的變化。再利用微電腦斷層 (Micro-CT) 及蘇木紫-伊紅染色 (H & E stain) 獲取股骨頭骨骺之骨形態學與組織學參數。由組織學切片結果顯示：藉由類固醇注射及負重訓練的方式大白鼠之骨骺處脂肪細胞有明顯體積增大與數量增加的趨勢，但沒有骨細胞死亡之組織學特徵，而在高頻率單一脈衝式電磁場刺激卻有破壞骨細胞的現象。由微電腦斷層 (Micro-CT) 結果顯示：使用 One-way ANOVA 進行 7 組 22 週犧牲之大鼠股骨骨骺 (epiphysis) 分析比較負控制組、正控制組、高頻率單一脈衝式電磁場組、低頻率單一脈衝式電磁場組骨骺 (epiphysis) 之骨體積百分比、骨小樑厚度及骨小樑數量有相似的數值且沒有顯著差異。負控制組、正控制組、高頻率單一脈衝式電磁場組、低頻率單一脈衝式電磁場組骨骺 (epiphysis) 之骨體積百分比及骨小樑厚度高於對照組，但骨小樑數量卻低於對照組；合併治療組骨骺 (epiphysis) 之骨體積百分比及骨小樑厚度高於其他組別，但骨骺 (epiphysis) 之骨小樑數量卻低於其他組別。

結論
本研究結果推估在本實驗中單獨以單一脈衝式電磁場作治療對骨骺 (epiphysis) 的骨質沒有太大的影響，所以在骨體積百分比、骨小樑厚度及骨小樑數量沒有太大的改變，但可降低骨小樑間隙；而在合併福善美 (alendronate) 對骨骺 (epiphysis) 有較大的骨質影響，可提高骨體積百分比、骨小樑厚度，亦可降低骨小樑間隙。

With the medical advances, corticosteroid is widely used in the treatment of many diseases (ie. Endocrine Disorders、Rheumatic Disorders、Dermatologic Diseases、Allergic States), which is the most common non-traumatic cause of osteonecrosis of the femoral head. In clinical, the corticosteroid treatment has most contributions, but patients also exposed to the risk of osteonecrosis. Despite the strong association of glucocorticoids with osteonecrosis of the femoral head, but the underlying mechanisms have been unclear. In Taiwan, the illness largely affects male population, especially the age in forty-five to sixty-four(1). It is initially asymptomatic, and if left untreated, more than 70 % of instances of osteonecrosis of the femoral head lead to collapse of the femoral head with subsequent hip joint destruction. Most cases advance to secondary osteoarthritis (OA), and the patient eventually requires total hip arthroplasty (THA). Therefore, early intervention before collapse would be a better strategy for treatment(2). The purpose of this study is using single pulsed electromagnetic field (SPEMF) and Alendronate on steroid-induced osteonecrosis femoral head of S.D. rats animal model. To hope finding a new therapeutic to treatment femoral head osteonecrosis.

Materials and Methods:
12-weeks-old S.D. rats were randomly assigned seven groups including control group (A), negative control group (B), positive control group (C), HF-SPEMF group (D), LF-SPEMF group (E), HF-SPEMF + ALN group (F) and LF-SPEMF + ALN group (G). 70 rats was injection of 40 mg/kg methylprednisolone every other day for two weeks and treadmill for four weeks. The rats were treatment using SPEMF and SPEMF combined with Alendronate for six weeks after the establishment of animal model with femoral head osteonecrosis.

Ruselt
After the rats were sacrificed, and femoral head were took out. The morphology were observed normal of each groups. Using H & E stain to observe epiphysis of the femoral head. The result were observed bone morrow, the rats injection steroids and treadmill training has hypertrophy and hyperplasia of fat cells on epiphysis, and most groups of the osteocyte was still alive, but in HF-SPEMF group, which osteocyte was death. Using Micro-CT scanning to analysis percent of bone volume, trabecular thickness, trabecular separation, and trabecular number and compare of seven groups using ANOVA analysis. The results of that negative control group, positive control group, HF-SPEMF group and LF-SPEMF group has similar and non-significant in percent of bone volume, trabecular thickness and trabecular number,. In the percent of bone volume and trabecular thickness, negative control group, positive control group, HF-SPEMF group and LF-SPEMF group was higher than control group, but the trabecular number opposite. In the percent of bone volume and trabecular thickness, HF-SPEMF + ALN group and LF-SPEMF + ALN group was higher than each groups, but the trabecular number opposite.

Conclusion
In this study estimate that using only SPEMF therapy was no effect in percent of bone volume, trabecular thickness and trabecular number on epophysis, but decrease trabecular separation. Combined with Alendronate make percent of bone volume, trabecular thickness and decrease trabecular separation.

中文摘要 Ⅰ
Abstract Ⅳ
目錄 Ⅶ
附表目錄 Ⅹ
附圖目錄 XI
附錄目錄 XII
縮寫表 XIII
第一章、緒論 1
1-1 股骨頭壞死 1
1-2 流行病學 2
1-3 骨骼系統 3
1-4 股骨頭壞死之致病因素 6
1-5 股骨頭壞死之臨床診斷與鑑別 9
1-5-1 微電腦斷層掃描 10
1-5-2 股骨頭壞死之鑑別 13
1-6 股骨頭壞死之臨床治療 14
1-6-1 非手術治療 15
1-6-1 手術治療 19
1-7 股骨頭壞死動物模型 21
1-8 研究動機與目的 23
第二章、材料與方法 25
2-1 實驗材料 25
2-1-1 動物 25
2-1-2 藥品 25
2-1-3 試劑 25
2-1-4 試劑套組 26
2-1-5 實驗器具 26
2-1-6 實驗儀器 26
2-2 實驗方法 27
2-2-1 誘導股骨頭壞死之動物模型 28
2-2-2 物理性刺激 SPEMF 與藥物 ALN 複合治療股骨頭壞死之動物 28
2-2-3 股骨頭之取樣 29
2-2-4 高解析度電腦斷層掃描分析 29
2-2-5 股骨頭之包埋與組織切片 30
2-2-6 組織染色之分析 31
第三章、結果 36
3-1 活體大鼠股骨頭 Micro-CT 分析 36
3-2 大鼠股骨頭外觀之觀察 37
3-3 體外大鼠股骨頭 Micro-CT 分析 37
3-4大鼠股骨頭 H & E 染色觀察組織型態 39
3-5免疫組織化學染色觀察成骨細胞與蝕骨細胞之分佈 40
3-6 TUNEL assay 觀察細胞凋亡現象 41
3-7利用免疫組織化學染色分析成骨細胞與蝕骨細胞的生物標誌 42
第四章、討論 43
4-1 大鼠之骨化程度 43
4-2 動物模型 43
4-3 股骨頭壞死動物模型之治療 44
4-4 類固醇誘導骨細胞凋亡 48
第五章、結論 49
參考文獻 50
附表 60
附圖 67
附錄 80

1.呂宗學、賴國安。95年度股骨頭壞死與人工髖關節置換手術流行病學調查計畫。醫學系公共衛生學科暨公共衛生研究所。2006。.
2.Kuroda Y, Akiyama H, Kawanabe K, et al. Treatment of experimental osteonecrosis of the hip in adult rabbits with a single local injection of recombinant human FGF-2 microspheres. J Bone Miner Metab. 2010;28(6):608-616.
3.Mont MA, Jones LC, Hungerford DS. Nontraumatic osteonecrosis of the femoral head: ten years later. J Bone Joint Surg Am. 2006;88(5):1117-1132.
4.Parsons SJ, Steele N, Osteonecrosis of the femoral head: Part 1—Aetiology, pathogenesis, investigation, classification. Current Orthopaedics. 2007;21,457–463.
5.Agarwala S, Jain D, Joshi VR, Sule A. Efficacy of alendronate, a bisphosphonate, in the treatment of AVN of the hip. A prospective open-label study. Rheumatology (Oxford). 2005;44(3):352-359.
6.Hofstaetter JG, Wang J, Yan J, Glimcher MJ. The effects of alendronate in the treatment of experimental osteonecrosis of the hip in adult rabbits. Osteoarthritis Cartilage. 2009;17(3):362-370.
7.Mont MA, Hungerford DS. Non-traumatic avascular necrosis of the femoral head. J Bone Joint Surg Am. 1995;77(3):459-474.
8.Mankin HJ. Nontraumatic necrosis of bone (osteonecrosis). N Engl J Med. 1992;326(22):1473-1479.
9.Assouline-Dayan Y, Chang C, Greenspan A, Shoenfeld Y, Gershwin ME. Pathogenesis and natural history of osteonecrosis. Semin Arthritis Rheum. 2002;32(2):94-124.
10.Kerachian MA, Séguin C, Harvey EJ. Glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action. J Steroid Biochem Mol Biol. 2009;114(3-5):121-128.
11.Koo KH, Kim R, Kim YS, et al. Risk period for developing osteonecrosis of the femoral head in patients on steroid treatment. Clin Rheumatol. 2002;21:299-303.
12.Ninomiya S, Nagasaka Y, Tagawa H. Slipped capital femoral epiphysis. A study of 68 cases in the eastern half area of Japan. Clin Orthop Relat Res. 1976;(119):172-176.
13.Kang JS, Park S, Song JH, Jung YY, Cho MR, Rhyu KH. Prevalence of osteonecrosis of the femoral head: a nationwide epidemiologic analysis in Korea. J Arthroplasty. 2009;24(8):1178-1183.
14.Fukushima W, Fujioka M, Kubo T, Tamakoshi A, Nagai M, Hirota Y. Nationwide epidemiologic survey of idiopathic osteonecrosis of the femoral head. Clin Orthop Relat Res. 2010;468(10):2715-2724.
15.Stein GS, Lian JB, Stein JL, Van Wijnen AJ, Montecino M. Transcriptional control of osteoblast growth and differentiation. Physiol Rev. 1996;76(2):593-629.
16.林齊宣、張丙龍。解剖學原理與實用。合記圖書出版社。1997。
17.賴家欣。臨床解剖學精華。合記出版社。2010。
18.行政院衛生署食品藥物管理局
19.Silverman SL, Lane NE. Glucocorticoid-induced osteoporosis. Curr Osteoporos Rep. 2009;7(1):23-26.
20.Gennari L, Bilezikian JP. Glucocorticoid-induced osteoporosis: hope on the HORIZON. Lancet. 2009;373(9671):1225-1226.
21.Felson DT, Anderson JJ. Across-study evaluation of association between steroid dose and bolus steroids and avascular necrosis of bone. Lancet. 1987;1:902-906.
22.Migliaresi S, Picillo U, Ambrosone L, et al. Avascular osteonecrosis in patients with SLE: relation to corticosteroid therapy and anticardiolipin antibodies. Lupus. 1994;3(1):37-41.
23.Wang GJ, Cui Q, Balian G. The pathogenesis and prevention of steroidinduced osteonecrosis. Clin Orthop Relat Res. 2000;370:295-310.
24. Inoue S, Horii M, Asano T, et al. Risk factors for nontraumatic osteonecrosis of the femoral head after renal transplantation. J Orthop Sci. 2003;8(6):751-756.
25.Griffith JF, Antonio GE, Kumta SM, et al. Osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids. Radiology. 2005;235:168-175.
26.Sakamoto M, Shimizu K, Iida S, Akita T, Moriya H, Nawata Y. Osteonecrosis of the femoral head: a prospective study with MRI. J Bone Joint Surg Br. 1997;79:213-219.
27.Fink B, Degenhardt S, Paselk C, Schneider T, Modder U, Ruther W. Early detection of avascular necrosis of the femoral head following renal transplantation. Arch Orthop Trauma Surg. 1997;116:151-156.
28.Hirota Y, Hirohata T, Fukuda K, et al. Association of alcohol intake, cigarette smoking, and occupational status with the risk of idiopathic osteonecrosis of the femoral head. Am J Epidemiol. 1993;137:530-538.
29.Matsuo K, Hirohata T, Sugioka Y, Ikeda M, Fukuda A. Influence of alcohol intake, cigarette smoking, and occupational status on idiopathic osteonecrosis of the femoral head. Clin Orthop Relat Res. 1988;234:115-123.
30.Glueck CJ, Fontaine RN, Gruppo R, et al. The plasminogen activator inhibitor-1 gene, hypofibrinolysis, and osteonecrosis. Clin Orthop Relat Res. 1999;366:133-146.
31.Liu YF, Chen WM, Lin YF, et al. Type II collagen gene variants and inherited osteonecrosis of the femoral head. N Engl J Med. 2005;352:2294-2301.
32.Miller KD, Masur H, Jones EC, et al. High prevalence of osteonecrosis of the femoral head in HIV-infected adults. Ann Intern Med. 2002;137:17-25.
33.Blacksin MF, Kloser PC, Simon J. Avascular necrosis of bone in human immunodeficiency virus infected patients. Clin Imaging. 1999;23:314-318.
34.Ries MD, Barcohana B, Davidson A, Jergesen HE, Paiement GD. Association between human immunodeficiency virus and osteonecrosis of the femoral head. J Arthroplasty. 2002;17:135-139.
35.Montella BJ, Nunley JA, Urbaniak JR. Osteonecrosis of the femoral head associated with pregnancy. A preliminary report. J Bone Joint Surg Am. 1999;81:790-798.
36.Koo KH, Kim R. Quantifying the extent of osteonecrosis of the femoral head. A new method using MRI. J Bone Joint Surg Br. 1995;77:875-880.
37.莊克士。醫學影像物理學。合記圖書出版社。1988。
38.網路資源http://www.szsbetter.com/ft/2-a.html
39.網路資源http://www.iplab.tcu.edu.tw/data/CT/CT_hi.htm
40.陳苡阡。股骨頭壞死的動物模型之建立。嘉南藥理科技大學生物科技系碩士論文。2009。
41.Arlet J, Ficat RP. Forage-biopsie de la tete femorale dans Ɩ’osteonecrose primative. Rev Rhumat. 1964;31:257–264.
42.Ficat RP, Arlet J. Necrosis of the femoral head. Williams & Wilkins. 1980;171-182.
43.Ficat, RP. Idiopathic bone necrosis of the femoral head. Early diagnosis and treatment. J Bone Joint Surg Br. 1985;67(1):3-9.
44.Steinberg ME, Hayken GD, Steinberg DR. A quantitative system for staging avascular necrosis. J Bone Joint Surg Br. 1995;77(1):34-41.
45.Kawamura K, Chung KC. Treatment of scaphoid fractures and nonunions. J Hand Surg Am. 2008;33(6):988-997.
46.Mont MA, Carbone JJ, Fairbank AC. Core decompression versus nonoperative management for osteonecrosis of the hip. Clin Orthop Relat Res. 1996;324:169-178.
47.Kasting GB, Francis MD. Retention of etidronate in human, dog, and rat. J Bone Miner Res.1992;7:513–522.
48.Seeman E. Understanding how antiresorptive agents optimize therapeutic effect. J Bone Miner Res. 2002;17(1):S125-541.
49.Lai KA, Shen WJ, Yang CY, Shao CJ, Hsu JT, Lin RM. The use of alendronate to prevent early collapse of the femoral head in patients with nontraumatic osteonecrosis. J Bone Joint Surg Am. 2005;87:2155-2159.
50.Pritchett JW. Statin therapy decreases the risk of osteonecrosis in patients receiving steroids. Clin Orthop Relat Res. 2001;386:173-178.
51.Xiu Z, Li M, Zhou S, Dou H, Zhou H, Chen C. A new method for the preparation of human parathyroid hormone 1-34 peptides. Biotechnol Appl Biochem. 2002;36(2):111-117.
52.董耀天。Alendronate與Teriparatide對股骨頭壞死之崩塌預防-大鼠動物實驗。嘉南藥理科技大學生物科技系碩士論文。2010。
53.Glueck CJ, Freiberg RA, Sieve L, Wang P. Enoxaparin prevents progression of stages I and II osteonecrosis of the hip. Clin Orthop Relat Res. 2005;435:164-170.
54.Ishida M, Fujioka M, Takahashi KA, Arai Y, Kubo T. Electromagnetic fields: a novel prophylaxis for steroid-induced osteonecrosis. Clin Orthop Relat Res. 2008;466(5):1068-1073.
55.Midura RJ, Ibiwoye MO, Powell KA, et al. Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies. J Orthop Res. 2005;23(5):1035-1046.
56.Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem. 1994;55(3):273-286.
57.Manolagas SC, Jilka RL. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med. 1995; 332:305-311.
58.Hronik-Tupaj M, Rice WL, Cronin-Golomb M, Kaplan DL, Georgakoudi I. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields. Biomed Eng Online. 2011;10:9.
59.Tsai MT, Li WJ, Tuan RS, Chang WH. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res. 2009;27(9):1169-1174.
60.Fu Y, Lin C, Chang J, Wang G, Ho M. A novel single pulsed electromagnetic field stimulates the osteogenesis of human bone marrow derived mesenchymal stem cells. J Bone . 2009;(44):S253–S338.
61.洪世彥。脈衝磁場刺激研發及對骨髓間葉細胞之骨化效應。國立成功大學醫學工程研究所碩士論文。2007。
62.林摯鈞。電磁場刺激對於人類間葉幹細胞之增殖與骨分化作用之探討。國立成功大學醫學工程研究所碩士論文。2007。
63.張昱婷。探討物理性刺激對於骨質疏鬆症之影響。國立成功大學醫學工程研究所碩士論文。2009。
64.Peskin B, Shupak A, Levin D, et al. Effects of non-weight bearing and hyperbaric oxygen therapy in vascular deprivation-induced osteonecrosis of the rat femoral head. Undersea Hyperb Med. 2001;28:187-194.
65.Reis ND, Schwartz O, Militianu D, et al. Hyperbaric oxygen therapy as a treatment for stage-I avascular necrosis of the femoral head. J Bone Joint Surg Br. 2003;85:371-375.
66.Lieberman JR, Conduah A, Urist MR. Treatment of osteonecrosis of the femoral head with core decompression and human bone morphogenetic protein. Clin Orthop Relat Res. 2004;429:139-145.
67.Sanjay BK, Moreau PG. Bipolar hip replacement in sickle cell disease. Int Orthop. 1996;20:222-226.
68.Cuckler JM, Moore KD, Estrada L. Outcome of hemiresurfacing in osteonecrosis of the femoral head. Clin Orthop Relat Res. 2004;429:146-150.
69.Takaoka K, Nishina T, Ohzono K, et al. Bipolar prosthetic replacement for the treatment of avascular necrosis of the femoral head. Clin Orthop Relat Res. 1992;277:121-127.
70.Yoshida T, Kanayama Y, Okamura M, Negoro N, Inoue T, Yoshikawa J. Longterm observation of avascular necrosis of the femoral head in systemic lupus erythematosus: an MRI study. Clin Exp Rheumatol. 2002;20:525-530.
71.Castro FP Jr, Barrack RL. Core decompression and conservative treatment for avascular necrosis of the femoral head: a meta-analysis. Am J Orthop. 2000; 29:187-194.
72.Rijnen WH, Gardeniers JW, Buma P, Yamano K, Slooff TJ, Schreurs BW. Treatment of femoral head osteonecrosis using bone impaction grafting. Clin Orthop Relat Res. 2003;417:74-83.
73.Urbaniak JR, Coogan PG, Gunneson EB, Nunley JA. Treatment of osteonecrosis of the femoral head with free vascularized fibular grafting. A long-term followup study of one hundred and three hips. J Bone Joint Surg Am. 1995;77:681-694.
74.Berend KR, Gunneson EE, Urbaniak JR. Free vascularized fibular grafting for the treatment of postcollapse osteonecrosis of the femoral head. J Bone Joint Surg Am. 2003;85:987-993.
75.Chan YS, Shih CH. Bipolar versus total hip arthroplasty for hip osteonecrosis in the same patient. Clin Orthop Relat Res. 2000;379:169-177.
76.Hernigou P, Bachir D, Galacteros F. Avascular necrosis of the femoral head in sickle-cell disease. Treatment of collapse by the injection of acrylic cement. J Bone Joint Surg Br. 1993;75:875-880.
77.Wood ML, McDowell CM, Kerstetter TL, Kelley SS. Open reduction and cementation for femoral head fracture secondary to avascular necrosis: preliminary report. Iowa Orthop J. 2000;20:17-23.
78.Adili A, Trousdale RT. Femoral head resurfacing for the treatment of osteonecrosis in the young patient. Clin Orthop Relat Res. 2003;417:93-101.
79.Yamano K, Atsumi T, Muraki M, Yoshihara S, Kajihara T. The blood supply of the lateral epiphyseal arteries in Perthes'' disease. J Bone Joint Surg Br. 2000;82(3):392-398.
80.Iwasaki K, Hirano T, Sagara K, Nishimura Y. Idiopathic necrosis of the femoral epiphyseal nucleus in rats. Clin Orthop Relat Res. 1992;277:31-40.
81.Conzemius MG, Brown TD, Zhang Y, Robinson RA. A new animal model of femoral head osteonecrosis: one that progresses to human-like mechanical failure. J Orthop Res. 2002;20(2):303-309.
82.Manggold J, Sergi C, Becker K, Lukoschek M, Simank HG. A new animal model of femoral head necrosis induced by intraosseous injection of ethanol. Lab Anim. 2002;36(2):173-180.
83.Yang L, Boyd K, Kaste SC, Kamdem Kamdem L, Rahija RJ, Relling MV. A mouse model for glucocorticoid-induced osteonecrosis: Effect of a steroid holiday. J Orthop Res. 2009;27(2):169-175.
84.Bekler H, Uygur AM, Gökçe A, Beyzadeoğlu T. The effect of steroid use on the pathogenesis of avascular necrosis of the femoral head: an animal model. Acta Orthop Traumatol Turc. 2007;41(1):58-63.
85.Iwamoto J, Shimamura C, Takeda T, et al. Effects of treadmill exercise on bone mass, bone metabolism, and calciotropic hormones in young growing rats. J Bone Miner Metab. 2004;22(1):26-31.
86.Tamaki H, Akamine T, Goshi N, Kurata H, Sakou T. Effects of exercise training and etidronate treatment on bone mineral density and trabecular bone in ovariectomized rats. Bone. 1998;23(2):147-153.
87.陳柏霖。探討物理性刺激應用於類固醇誘發股骨頭骨質改變之影響。國立成功大學醫學工程研究所碩士論文。2011。