骨骼是提供人體支撐、保護和運動功能的重要器官。當壓砸型式的外力造成骨折時，常合併血管破裂和軟組織腫脹，這些因素會導致骨組織處於缺血及低氧狀態，引起發炎、水腫，最後形成骨壞死。骨癒合過程可分為發炎、軟性骨痂生成、硬性骨痂生成以及組織重塑四個階段；適度的發炎反應已被證實有益於骨組織的修復，發炎反應主要是由花生四烯油酸(arachidonic acid, AA)藉由環氧合酶(cyclo-oxygenase, COX)或5-脂氧合酶(5-lipo-oxygenase, 5-LOX)作用，轉換成前列腺素(prostaglandin, PG)及白三烯(leukotrienes, LT)產生，而cyclo-oxygenase -2 (COX-2)是被骨折誘導的發炎反應因子之一，經由COX-2調控釋放prostaglandin (PG)來控制蝕骨細胞的骨吸收作用及成骨細胞的骨新生成作用。另外骨折後的疼痛是臨床上必需迅速解決的重要問題，非類固醇抗發炎藥則是現在臨床上最常使用的止痛劑。目前關於低氧環境對成骨細胞的傷害，和低氧環境下，非類固醇抗發炎藥對成骨細胞凋亡及骨癒合的影響都還不是十分明瞭。因此本研究的目的在於探討低氧環境對成骨細胞的傷害和低氧環境下，非類固醇抗發炎藥對成骨細胞凋亡及骨癒合的影響。
為了瞭解低氧環境對人類成骨細胞凋亡及骨癒合的影響，我們先建構一個在低氧狀態下人類成骨細胞的凋亡模型，利用氯化鈷(COCl2)和Desferrioxamine兩種低氧模擬藥，加入人類成骨細胞(MG-63和hFOB)中，分別在0、4、8、12、24、48小時後，觀察細胞數目和形態、缺氧誘發因子(hypoxia inducible factor -1α, HIF-1α)、COX-2和cleaved poly ADP-ribose polymerase (PARP)的變化。另外在低氧狀態下，於人類成骨細胞加入四種抗發炎藥: 三種非類固醇抗發炎藥indomethacin(IND)、sulindac(SUL)、aspirin(Asp)和另一類抗發炎藥salicylate，在加藥1小時後觀察細胞數目、COX-2 、Caspase-3、PARP cleavage和鹼性磷酸酶(alkaline phosphatase, ALP)的變化。其次是將6-8週大的B6母小鼠麻醉，先以直徑0.25mm不銹鋼釘由左膝部穿入脛骨髓腔，再利用自由落體撞擊損傷模擬裝置，給予5.15×10-1牛頓的垂直撞擊力量，造成脛骨骨折，分別於骨折後2天、1週、2週、8週犧牲小鼠，取下脛骨骨折區域之組織做H&E染色，並利用微電腦斷層在18μm/75kV/112μA和0.025mm鈦濾片條件下，於骨折後1、2、4、8週分析骨體積(bone volume)、骨痂體積(callus volume)、組織體積(total tissue volume)和骨鈣化密度(bone mineralization density)的變化。另外骨折後連續給予口服7日之indomethacin (100mg /50kg/day)，於第2天、2週、4週分別做Hematoxylin and Eocin(H&E)、COX-2 immuno-histo-chemistry和Terminal deoxy- nucleotidyl transferase dUTP nick end-labeling(TUNEL)染色，以瞭解indomethacin對成骨細胞凋亡的影響。骨折後連續給予7日之口服阿斯匹靈(Asp)、非選擇性COX抗發炎藥-indomethacin、選擇性COX-2抗發炎藥-SC-236，來研究非類固醇抗發炎藥物對早期骨癒合的影響。
結果顯示，氯化鈷(COCl2)和Desferrioxamine確實在不同的時間點會造成人類成骨細胞數目減少，低氧誘發因子(HIF-1α) 和COX-2的表現增加。加入藥物48小時後cleaved PARP的增加和Annexin-V陽性/Propidium iodide陰性染色則證明成骨細胞有凋亡的現象。氯化鈷(COCl2)和Desferrioxamine都會抑制鹼性磷酸酶(ALP)功能。在低氧狀態下，IND 和SUL使得cleaved PARP表現增加，顯示都會促進人類成骨細胞的凋亡，也幾乎完全抑制鹼性磷酸酶(ALP)的作用。而阿斯匹靈(Asp)則沒有明顯促進成骨細胞凋亡的現象。在骨折後第1、2、4和8週利用微電腦斷層掃描骨頭癒合情況、計算骨痂體積、骨頭體積、組織體積和骨鈣化密度；骨修復過程中，在第2週有最大的骨痂體積，有最大骨體積在第4週，在第8週有最高的骨鈣化密度表現。TUNEL染色顯示骨折後給予IND會增加成骨細胞的凋亡和在第4週時造成骨癒合的延緩。IND會降低LTB4受體(BLT1)但並沒有明顯改變細胞凋亡和COX-2的表現量。阿斯匹靈(Asp)對骨折癒合的影響在較早的軟骨細胞形成階段，同時也減少了細胞凋亡和降低COX-2及BLT1的表現量。SC-236降低COX-2的表現量但不影響細胞凋亡和BLT1的表現量。
根據上述的實驗結果，我們認為低氧狀態和非類固醇抗發炎藥物(尤其是IND)確實會使得人類成骨細胞凋亡，並可能延緩骨癒合。我們的發現可以研究不同的治療策略，協助抗發炎藥物的選擇，及提供骨折患者較佳的臨床治療方式。
關鍵字: 缺氧、成骨細胞、凋亡、發炎、非類固醇抗發炎藥物、骨折癒合

The human skeleton provides mechanical support for weight bearing, protection and locomotion. In addition to bone fracture by direct external force, impairment of blood supply due to vessel damage and soft tissue swelling often combine simultaneously and result in ischemia-hypoxic injury of bone tissues, especially caused by crush injury. All together, these factors lead to the consequential bone tissue inflammation, edema and finally, necrosis. Pain is also an important symptom, clinically in need of solving immediately after injury. Currently, the most widely used analgesic drugs in orthopedic clinics are nonsteroidal anti-inflammatory drugs (NASIDs). However, the underlying molecular mechanism of hypoxia-induced bone injury and outcome for usage of anti-inflammatory drugs (AIDs) on fracture repair in hypoxic environment are still poorly understood.
To investigate the damage and functional deficiency of osteoblast in hypoxia, two hypoxia mimetics, cobalt chloride (CoCl2) and desferrioxamine (DFO), were used to create an in vitro hypoxic microenvironment in human osteoblast-like cells, MG-63 and hFOB. The cell damage was observed by cell number and morphology, hypoxia-inducible factor-1α (HIF-1α), cyclo-oxygenase-2 (COX-2) and cleaved poly ADP-ribose polymerase (PARP). Cell apoptosis was confirmed by WST-1 cytotoxic assays and flow cytometry. To simulate the use of NSAID after hypoxic injury, three kinds of NSAIDs, indomethacin (IND), sulindac sulfide (SUL), and aspirin (Asp), and sodium salicylate (NaS) were applied to osteoblasts subjected to hypoxia condition. Cyclo-oxygenase-2 (COX-2), caspase-3, PARP cleavage and alkaline phosphatase (ALP) were observed 48 hours after AIDs treatment.
Then, we create an animal model to evaluate effects of NSAIDs on healing of crush fracture. Tibia fracture of mouse was created by a free-dropping impacted-injury simulator and micro-computed tomography (μCT), was used to analyse bone volume, callus volume, total tissue volume and bone mineralization density. Indomethcin was continuously given by oral feeding for 7 days and the fracture area was harvested for H&E stain, immuno-histo-chemistry of COX-2 and TUNEL assay. The anti-inflammatory effects of NSAID treatments were investigated by providing a daily oral intake of Asp, IND, and a selective COX-2 inhibitor (SC-236) in mice after fracture injury. The inflammatory responses on fractured site with various NSAIDs treatments were identified by histological analysis.
The results showed that hypoxia reduced the cell number of osteoblasts and increased the expression of hypoxia-inducible factor-1α (HIF-1α) and cyclo-oxygenase-2 (COX-2) at different temporal characteristic among CoCl2 and DFO. The increase of cell apoptosis was confirmed by cleaved poly ADP-ribose polymerase (PARP) and assessed using flow cytometry by positive Annexin V / negative staining with propidium iodide. The functional expression of osteoblast in alkaline phosphatase (ALP) activity was significantly decreased by CoCl2 and inhibited when treated with DFO. The treatment of IND and SUL increased the percentage of cells apoptosis and showed higher expression in cleavage of PARP. The ALP activity was totally abolished in hypoxic osteoblasts with IND treatment. On the other hand, the cell damage and ALP function seemed unaffected by Asp treatment. Facilitation of osteoblast apoptosis occurred regardless of IND dosage under hypoxic condition. The highest volume of bone formation was detected at 4 weeks and highest callus volume can be identified as early as on 2 weeks. The highest mineral density in fracture bone was observed after 8 weeks of injury. IND-induced osteoblast apoptosis was confirmed by positive staining of TUNEL assay in fractured mice. The IND-treated fractured mice showed significant delay of fracture healing in bone tissue, and decreased expression of BLT1, but showed no significant changes in cell apoptosis or COX-2 expression comparing to fracture mice not receiving IND treatment. The Asp treatment significantly inhibited cell apoptosis, expression of COX-2 and BLT1. Similarly, treatment with SC-236 specifically reduced COX-2 expression in the tissue of fracture mice, but did not alter cell apoptosis or BLT1 expression.
According to the data gained from above, we believe that both hypoxia and NSAIDs induce osteoblast apoptosis. NSAIDs impair the healing of the fractured bone under hypoxia. These results may provide very valuable information in choosing the NSAID for patients with bone fracture. Key words: hypoxia；osteoblast；apoptosis；NSAID；fracture healing

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