臺灣博碩士論文加值系統

希爾-薩克斯病變主要發生於肩關節 ( Glenohumeral Joint ) 不穩定之球窩結構 ( Ball-and-Socket Structure ) 而前脫臼 (Anterior Dislocation) 所致。由於肩盂腔 ( Glenoid Cavity ) 及肱骨頭 ( Humeral Head ) 之間尺寸不匹配，導致不管在哪個旋轉角度總是只有三分之一之肱骨頭被肩盂腔所覆蓋，因此造成肩膀之先天性不穩定。希爾-薩克斯缺陷係當不穩定結構受到外力撞擊造成脫臼時，導致盂唇撕裂而長期使關節間相互碰撞而產生。由前人研究中可知，希爾-薩克斯缺陷會導致旋轉面被限制約30-40%之運動範圍，並且有極高之肩關節脫臼復發機率。
習慣性肩關節脫臼患者，在長期關節相互撞擊下將遭受劇烈疼痛與結構不穩定之問題，因此長期以來大尺寸之缺陷即須考慮接受關節置換手術。對於較小缺陷在臨床上已有自體移植手術與降落傘縫法等治療方式，由於其可用性與生物相容性不足常限制其臨床療效。
硫酸鈣（CS）早於1892年即備受矚目並使用於臨床治療上，故能確定其為人體內可運用之骨填充材料。然而本實驗之半水硫酸鈣已被證實其具有骨誘導以及骨傳導之生物特性，其卻從未被應用於治療希爾-薩克斯缺陷。因此，本研究利用生物良好性且可流動性之半水硫酸鈣注射於希爾-薩克斯缺陷，並使其缺陷藉由材料之刺激後進行骨組織修復。為使半水硫酸鈣於材料固化後機械性質之強化，於本實驗中結合硬化劑與其參與反應，並藉由黏著劑以提升材料與骨缺陷接合之強度。本研究分為三個章節以討論注射式之半水硫酸鈣結合黏著劑對希爾-薩克斯組織之修復情形，包括材料特性、體外生物相容性以及動物模型設計與其體內實驗。


第一章節:材料特性
半水硫酸鈣之製備係透過熱差分析儀（DSC）與熱重分析儀（TGA）進行熱處理條件評估。於熱差分析儀結果中發現二水硫酸鈣於120-140 時出現第一吸熱峰ΔH=713.54 J/g，意味著二水將於此溫度範圍下轉換為半水。並且於140-150℃時出現第二吸熱峰 ΔH=144.54 J/g，即於此溫度範圍將使半水轉換為無水。此外，由熱重分析儀結果中顯示，於119.5℃後二水將快速的轉換為半水。因此，本實驗將二水硫酸鈣置於5 kg/cm2之環境壓力下並進行9小時127℃之熱處理使其轉換為半水硫酸鈣。為了進一步確認二水硫酸鈣成功的轉換為半水硫酸鈣，係透過繞射分析儀（XRD）分析結晶型態，並且使用JCPDS資料系統進行比對。二水硫酸鈣之主晶面分別位於（020）、（021）和（041），並於九小時之熱處理後主晶面轉換為（010）、（310）和（202）之半水硫酸鈣結晶結構。由於繞射分析儀仍無法具體證明半水硫酸鈣之結晶型態（β或α相），因此藉由掃描式電子顯微鏡（SEM）進一步鑑別晶體之形成。二水硫酸鈣之微結構長度為163.06±11μm和寬為28.45±6.53μm之較寬針狀結構（N= 5），相較於蒸發過後之半水硫酸鈣為長為92.02±63μm和寬為17.31±2.4μm之較細短之針狀結構（N = 5）。
此外，半水硫酸鈣固化後之機械性質測試系統皆比照ASTM451-99a之測試規範。由結果中顯示，藉由氯化氫固化之半水硫酸鈣能有效透過離子鍵鍵結，並相較於半水硫酸鈣透過二次去離子水之氫鍵固化由5.39±0.42MPa提升至9.62±2.05MPa（N = 6）之抗壓強度。另一方面而言，於擠出測試中顯示出其兩者混合液與半水硫酸鈣固化之硬化時間皆約為10-14分鐘（N =6）。由統計分析後發現，其兩者並無顯著性差異且皆為臨床手術下可接受之硬化時間。
其兩種混合液使半水硫酸鈣固化後對其兩組材料進行降解率測試，於49天後其兩組固化後材料之降解率皆約達25％之重量損失與其pH值約落於5.5-6.0，並於材料外觀上並無明顯改變。由49天後之元素分析中顯示出，半水硫酸鈣結合氯化氫混合液之組別具有更強之化學鍵結可有效控制鈣離子之釋放。對於固化後材料孔隙率而言，半水硫酸鈣透過氫鍵結合之固化材料之微觀結構是具有互鎖結構，並擁有58％ 之孔隙率（N =10）。而半水硫酸鈣結合氯化氫之固化材料於顯微鏡下顯示出膠水狀結構，並其孔隙率為47％（N =10）。半水硫酸鈣不管透過氫鍵或離子鍵之鍵結其孔徑大小皆落於10-100μm，其有足夠空間使細遷入與貼附。

第二章節：體外實驗
本研究按照ISO-10993-5之規範藉由人類骨肉瘤細胞MG63來測試半水硫酸鈣結合二次去離子水、半水硫酸鈣結合氯化氫以及15％藻酸之生物相容性。將半水硫酸鈣結合二次去離子之粉體與半水硫酸鈣結合氯化氫之粉體分別融入培養液中直至飽和濃度，並其培養液中內含有10％牛血清與1％青黴素，接著分別與5× 之細胞數共培養 (N=5)。藻酸之生物毒性係將其0.1CC之膠態塗佈於24孔培養皿中並與5× 之細胞數共培養。於共培養1、3、5、7天後藉由酵素反應劑與細胞反應並置於酵素結合免疫吸附分析儀中進行細胞活性分析來驗證材料之生物相容性。經由實驗結果中發現，三種材料與無參與材料之控制組比較上並無統計上之明顯差異。（P〉 0.05）
本研究之細胞貼附實驗係仿照ASTM F813-83之規範設計，將半水硫酸鈣共結合二次去離子水與半水硫酸鈣結合氯化氫之固化材料置於24孔培養皿中並將5× 之細胞數種置材料表面，接著於1、3、5天後觀察細胞貼附情況。材料後處理以10％之多聚甲醛固定並以濃度梯度之酒精進行乾燥、鍍金後最後藉由電子顯微鏡進行觀察。在顯微鏡觀察下顯示出細胞於第一天皆有效的貼附於兩組材料表面，而於第五天後細胞明顯之遍布於材料表面上，並有大量分泌物。因此，我們將材料裁成兩半並觀察細胞遷移之情況，由顯微鏡中發現這些細胞於第5天已經成功之遷移置材料內部。
材料與骨頭之貼附強度係藉由豬之肱骨頭進行測試，其中沒有塗附藻酸之固化材料置於Hank氏溶液5分鐘後失去貼附能力並由骨頭中掉出。相較之下有塗附藻酸之固化材料置於5分鐘後仍有效之貼附於骨缺陷中，並其邊界強度可達到1.73±0.26 MPa（N = 6）。

第三章節：體內實驗
希爾-薩克斯之動物模型係建立於兔子之肱骨頭，並透過斷層掃描與組織型態分析而定義。其缺陷尺寸為肱骨之25％，直徑為7.5mm 與深度5mm。由於缺陷尺寸過深，於手術過程中出現滲血情形導致材料無法有效與周邊組織接合。因此於手術後兩周，再進行第二次手術將材料注入生物體內。手術過程中將半水硫酸鈣與氯化氫以1：1之比例於常溫下進行60秒之混合，其材料固化時間為10至11分鐘，並將0.1cc之15％藻酸同時注入生物體內。並於手術4週與12週後對其進行骨組織型態分析。
於斷層掃瞄分析中，可由2D影像看出手術4週後骨組織由缺陷邊界開始生長，並顯示殘留材料仍存於缺陷內。相較而言，術後4週之未添加材料之骨缺陷則無表現出組織修復情況，並由統計分析中顯示出兩者有顯著性之差異（P〈0.05）。由2D影像中可看出術後12週之骨缺陷已有明顯修復並且已無殘留材料之表現。然而，3D組織重建下之骨缺陷外表已於4週與12週後表現出良好修復情況。經由BV / TV值之計算下發現，不管是4週或12週之修復皆與缺陷組有統計上之明顯差異（P〈0.05），並且材料組於12週後與控制組之間沒有顯著性差異（P〉 0.05）。
為了進一步證實骨組織再生，應用4μm之石蠟組織切片並使用蘇木素-伊紅和Masson trichrome染劑進行組織觀察。於第4週組織染色結果中可看出骨頭已由邊界開始成長，並且有大量之成骨細胞已遷移至材料中以及顯示出結締組織之生成。然而，未添加材料之缺陷組於4週後完全沒有顯示出組織之形成。此外，材料組於手術12週後新生骨已完整取代材料並有良好之組織修復。然而，可由組織染色中觀察出缺陷組於12週後骨組織有100-700μm之組織再生並有結締組織形態呈現。
本研究之半水硫酸鈣/氯化氫/藻酸係提供簡單配製且擁有良好生物相容性之可注射式材料，並表現出材料應用於生物體內之可行性。因此，本篇研究可證實出半水硫酸鈣/氯化氫/藻酸之注射式材料係有應用於臨床上修復希爾-薩克斯缺陷之潛力。

The Hill-Sachs lesion is usually caused by shoulder dislocation at glenohumeral joint which is a ball and socket structure, and the dimension of humeral head is larger than glenoid fossa which makes it unstable and easily being dislocated. The dislocation causes the superior labrum anterior to posterior lesion and impinges into the bone, making engaging Hill-Sachs defect that is participated for the range of motion (ROM) in the 30% to 40% circumscribed. The Hill-Sachs lesion has been reported in high possibility of recurrent anterior dislocations.
When the instability of the shoulder gets worse caused by Hill-Sachs defect, the patient will suffer severe pain and the defect should consider surgical repair. Several autologous grafts and synthetic materials have been used as graft material; however, their availability and biocompatibility limit their clinical outcome. Thus, this study investigates to treat the Hill-Sachs lesion with injectable alpha Calcium Sulfate Hemihydrate (αCSH).
It is possible to be used inside the human body as bone filler. Virtually, the αCSH has been noticed having the characteristics of the osteo-induction and osteo-conduction in vivo; however, it has never been applied as bone filler to repair the Hill-Sachs defect. This study tests the possibility of using αCSH to repair the Hill-Sachs defect and was divided into three parts including αCSH characterization, in vitro biocompatibility of αCSH and in vivo Hill-Sachs model in rabbits:

Part 1 Material preparation:
The preparation of αCSH was from heat treatment of Calcium Sulfate Dihydrate (CSD, J.T. Baker) and the heat treatment conditions were evaluated by Differential Scanning Calorimetry (DSC) and Thermogravimetry Analysis (TGA). The result of DSC curve had first endothermic peak (△H=713.54 J/g) at 120-140℃ which means 2H2O converts to 1/2H2O, and there is the second endothermic peak (△H=144.54 J/g) at 140-150℃ for 1/2H2O to non-aqueous. Also, the TGA curve of CSD and CSH shows the decreasing in weight around 119.5℃. Combined the DSC and TGA results we defined the heat treatment temperature at 127℃ under 5kg/cm2 pressure for 9 hours. In the results of X-ray Diffraction (XRD) which match with JCPDS card and Jade system were to identify the completeness of phase transformation. The primary crystal planes of CSD were located at (020), (021), and (041). After 9 hours heat treatment the crystal planes of CSD were convert to αCSH at (010), (310), and (202). In spite of that the XRD still cannot specific determine the kind of crystallize (α or β phase), the microstructure from scanning electron microscopy (SEM) was used to further identify the crystal formation. In the SEM images of CSD with wider arrangement which the particles size were 163.06±11μm in length and 28.45±6.53μm in width (N=5). The microstructure of αCSH were with needle-shaped which particles size were 92.02±63μm in length and 17.31±2.4μm in width (N=5), from H2O evaporation.
In addition, the compressive strength of αCSH was tested by Materials Test Systems by following the ASTM 451-99a. The compressive strength of αCSH hardening with DD water was at 5.39±0.42MPa (N=6) and the αCSH with curing agent was enhanced the compressive stress to 9.62±2.05MPa (N=6). However, the curing time of αCSH/DD water and the αCSH/HCl were with no significant difference at 10-14 minutes and with proper hardening time for surgery. (N=6)
Besides, the degradation of materials was with similar degradation rate about 25% weight loss after 49 days and the pH value at 5.5-6.0. However the αCSH/ HCl group has a stronger chemical bounding which can effectively control the release of calcium ions in the results of EDS after 49 days. All of the scaffolds had no significant change for the appearance after 49 days degradation. Therefore, the microstructure of hardening material of αCSH/ DD water has an interlocking structure with 58% of porosity (N=10). However, the material of αCSH/HCl demonstrated the glue-like structure with 47% of porosity (N=10). Both materials have a pore size of 10-100 μm which is adequate dimension for cell migration and attachment.

Part 2 in vitro test:
A human osteoblast-like cell line, MG63 (Homo sapiens bone osteosarcoma), was used to test the biocompatibility of αCSH/DD water, αCSH/HCl, and 15% alginate and followed by ISO-10993-5. The cytotoxicity of αCSH/DD water and αCSH/HCl group which co-cultured with 5×104 cells in 24-well plates in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% Fetal Bovine Serum, 1% penicillin-streptomycin and saturation concentration of material powder. The alginate was coated with 0.1 cc material on the 24-well plates and co-cultured with 5×104 cells. After the incubation of cells with materials for 1, 3, 5, and7 days, absorbance was set at 490 nm to evaluate the cell viability in response to the cytotoxicity of materials. The results of MTS assay shows the co-cultured cells with materials had no significant difference of cytotoxicity compared with control group. (P〉0.05)
The cell attachments of materials were followed by ASTM F813-83. The scaffolds were seeded with 5×104 cells on the surface and incubated in 24 well plates, observed at 1, 3, 5 days. They were dehydrated in a graded series of ethanol, treated with 10% paraformaldehyde, coated with gold, and observed by SEM. In the results of microscopy demonstrated that cells were extended and attached on the scaffolds successfully after 1 day. Cells were spread all over the surface of scaffolds and with plenty of secretion, after 5 days. The SEM shows the cells had migrated into the cut in halve scaffold at day 5.
In bounding test, the humeral heads of pork were used in the push out test. The material without alginate drops out from the bone defect after immersing in Hank’s solution for 5 minutes. However, the material with alginate was effectively attached on the bone defect and the bounding force had reached to 1.73±0.26 MPa (N=6)

Part 3 in vivo test:
The Hill-Sachs model was built by creating a defect on rabbit’s humeral head. The size and location of defect to mimic the Hill-Sachs defect was verified by histology and micro-CT after sacrificed. Defect size was 25% of humeral head and the diameter was 7.5mm and the depth was 5mm. The deflection depth was too deep which destroy the bone marrow and cause angiostasis which lead to a failure of attaching the materials into the bone defects. Therefore, the materials were injected two weeks after the surgery.
The process of mixing αCSH and hardening solution are1:1 ratio at room temperature with stirring for 60 seconds then it was injected with 15% alginate.αCSH hardening reaction took 10 to 11 minutes of curing time at body temperature (37 °C) The functionality of repairing Hill-Sachs defect after αCSH injection were evaluated by radiography and histology at 4 and 12 weeks postoperatively.
CT analysis shows, the bone remodeling had tissue regeneration starting from the edge of defect and the residue of materials were showed on the 2D image after 4 weeks. In contrast, the defect without treatment had no obvious tissue repaired on the 2D image after 4 weeks. However, the material had been completely replaced by the newly bone after 12 weeks. In the result of 3D reorganization, the bone defect had well appearance after 4 and 12weeks (N=6). The value of BV/TV showed that the material groups had obvious difference with defect groups (P〈0.05). Furthermore, there were no significant difference compare with defect group treated with materials after 12 weeks and the control group. (P〉0.05)
The histology applied with HE and Masson trichrome staining by paraffin sections for 4μm was use to confirm the bone regeneration. In the results of histology, the bone repaired started from the edge, the plenty of osteoblasts had migrated into the material and the connective tissue showed the connection between newly bone and the material after 4 weeks. However, the defect group had no tissue regeneration on histology after 4 weeks. Besides, the bone regeneration of material group has been completed, comparing with the defect group which was 100-700μm regenerated after 12 weeks.
The αCSH/HCl/Alginate provided a simple preparation of injectable performance with great biocompatibility in vitro and bone repaired, which has a potential in repairing the Hill-Sachs defect surgery.

Table of Content
中文摘要 I
Abstract VI
Acknowledgement XI
Table of Content XIII
List of Figures XVIII
List of Tables XXIV
Chapter 1 Introduction 1
1.1 Structure, composition and biomechanics of humeral head 1
1.2 Hill-Sachs lesion and the probability of occurrence 4
1.3 Current treatments of Hill-Sachs defect repair 8
1.3.1 Allograft 9
1.3.2 Autograft 9
1.3.3 Remplissage 10
1.3.4 Arthroplasty 10
1.4 Mechanism of bone remodeling 13
1.4.1 Osteoblast 15
1.4.2 Osteoclast 16
1.4.3 Bone remodeling 17
1.5 The bone cement 20
1.5.1 Polymer bone cement 23
1.5.2 Bioactive ceramics 23
1.5.3 Calcium sulfate hemihydrate 24
1.5.4 Mechanism of calcium-sensing receptor in bone remodeling 28
1.6 Alginate 29
Chapter 2 Objective 35
2.1 Part I (Develop a bilayer injectable material) 35
2.2 Part II (in vitro/ex vivo study) 35
2.3 Part III (Rabbit animal model set up and bone regeneration of Hill-Sachs defect) 35
Chapter 3 Materials and Methods 36
3.1 Drugs of experiment 36
3.2 Flow chart of experimental 37
3.3 Part I- Material preparation 38
3.3.1 Flow chart of material preparation 38
3.3.2 Procedure of calcium sulfate hemihydrate 38
3.3.3 Characteristics of materials 40
3.3.3.1 X-ray Diffraction (XRD) 40
3.3.3.2 Scanning Electron Microscopy (SEM) 41
3.3.3.3 Energy Dispersive Spectroscopy (EDS) 43
3.3.3.4 Ion Sputter Coater 43
3.3.4 Thermal properties of material 44
3.3.4.1 Differential Scanning Calorimetry (DSC) 44
3.3.4.2 Thermogravimetry Analysis (TGA) 45
3.3.5 Mechanical properties of material 46
3.3.5.1 Vicat needle 46
3.3.5.2 Mechanical Testing System 47
3.3.5.3 Extrusion test 49
3.3.6 Viscosity of alginate 50
3.4 Part II-in vitro/ ex vivo 51
3.4.1 Flow chart of in vitro and ex vivo test 51
3.4.2 Physiological of material 51
3.4.2.1 Degradation of material 51
3.4.2.2 Porosity 52
3.4.3 Cell maintain and biocompatibility 53
3.4.3.1 Cell maintain 53
3.4.3.2 Cell viability assay 53
3.4.3.3 Cell attachment 54
3.4.4 ex vivo test 54
3.4.4.1 Boundary test 54
3.5 Part III-in vivo test 56
3.5.1 Flow chart of in vivo test 56
3.5.2 Surgery procedure 57
3.5.3 Create defects 57
3.5.4 Material injection 59
3.5.5 Micro-Computed Tomography (Micro-CT) 61
3.5.6 Histology 61
3.5.6.1 Hematoxylin and Eosin stain 62
3.5.6.2 Masson’s Trichrome stain 63
Chapter 4 Results 65
4.1 Part I 65
4.1.1 Characteristics of materials 65
4.1.1.1 Results of X-ray Diffraction 65
4.1.1.2 Results of SEM 66
4.1.1.3 EDS analysis 69
4.1.2 Thermal properties 69
4.1.2.1 DSC and TGA analysis 69
4.1.3 Mechanical properties 71
4.1.3.1 Vicat needle 71
4.1.3.2 MTS analysis 71
4.1.3.3 Extrusion test 73
4.1.4 Alginate concentration 73
4.1.5 Degradation 74
4.1.6 Porosity 80
4.2 Part II in vitro/ ex vivo 80
4.2.1 Biocompatibility 80
4.2.1.1 MTS assay 80
4.2.1.2 Cell attachment 83
4.2.2 ex vivo 89
4.2.2.1 Boundary test 89
4.3 Part III in vivo 89
4.3.1 CT analysis 89
4.3.2 Mimics reorganization 93
4.3.3 Histology 95
4.3.3.1 HE stain 95
4.3.3.2 Masson’s Trichrome stain 101
Chapter 5 Discussion 107
5.1 Part I material preparation 107
5.2 Part II in vitro/ ex vivo test 111
5.3 Part III in vivo test 113
Chapter 6 Conclusions 121
6.1 Part I material preparation 121
6.2 Part II in vitro/ ex vivo test 121
6.3 Part III in vivo test 121
Chapter 7 Limitation 123
Reference 124


List of Figures
Figure 1 Structure of humerus(David) 1
Figure 2 Structure of humergleniod joint(HoustonMethodist, 2003) 2
Figure 3 Structure of clavicle(Haghighat) 3
Figure 4 The anatomy structure of AC, SC, GH, and ST joints in shoulder(P.B., 2009) 4
Figure 5 The occurrence of Hill-Sachs lesion. 5
Figure 6 The distances from the medial margin of the contact area to the edge of the articular surface of the humeral head (MA) and to the medial margin of the cuff attachment site on the greater tuberosity (MF) were measured. (C) Articular center of the humoral head; (M) most medial point; (A) lateral margin of articular surface; (F) footprint. (Yamamoto, et al., 2007) 7
Figure 7 (A) Coracoid graft is fixed to glenoid with 2 bicortical screws. If need be, the graft can be contoured with a power burr to fit the curve of the anterior-inferior glenoid. (B) Note how the coracoid graft restores the pear shape of the glenoid by widening its inferior diameter.(Burkhart ＆ De Beer, 2000) 8
Figure 8 Three Hill-Sachs reconstructions. (A) A remplissage procedure with placement of suture anchors in the trough of a 30% Hill-Sachs defect. (B) The remplissage sutures are passed through the infraspinatus, running from superior to inferior, to draw soft tissue into the entire length of defect. (C) The allograft humeral head reconstruction of the 30% Hill-Sachs lesion, viewed from medial to lateral, is held in place by standard cancellous bone screws. (D) The allograft viewed from the inferomedial aspect of humeral head demonstrates reestablished congruity of articular surface. (E) The partial resurfacing HemiCap Arthrosurface component placed in a 30% Hill-Sachs defect. (F) The partial resurfacing component viewed from inferomedial aspect of humeral head demonstrates re-established congruity of the articular surface. (Giles et al., 2012) 11
Figure 9 Structure of compact bone and spongy bone.(Rogóż, 2010) 14
Figure 10 The compositions of bone("Orthopaedic Surgery/Histology of Bone," 2009) 15
Figure 11 The proposed model of coordinated regulation of osteoblast differentiation and proliferation during bone formation by Osx and Wnt/β-catenin signaling.(Tang, et al., 2012) 16
Figure 12 The function of osteoclast 17
Figure 13 Bone remodeling procedure(Olivier Drevelle, 2013) 20
Figure 14 The heat treatment temperature of calcium sulfate hemihydrate (Singh ＆ Middendorf, 2007) 25
Figure 15 Feedback loops in calcium ion (Ca2+) and phosphate (P) homeostasis (DeLuca, 1986; Schjeide, 1985; Trechsel, 1986) 29
Figure 16 The block G monomer interact with calcium(Lersch, 2006) 33
Figure 17 The compose of alginate(McHugh) 33
Figure 18 The mechanisms of alginate combine with calcium 34
Figure 19 Flow chart of the experiment 37
Figure 20 Flow chart of material preparation 38
Figure 21 Autoclave 39
Figure 22 Principle of XRD 41
Figure 23 Scatter of electron beam 42
Figure 24 Scanning Electron Microscopy 43
Figure 25 Ion Sputter Coater 44
Figure 26 Differential Scanning Calorimetry 45
Figure 27 Thermogravimetry Analysis 46
Figure 28 Vicat needle 47
Figure 29 MTS testing 48
Figure 30 Shaping chamber 48
Figure 31 Samples of MTS 49
Figure 32 Extrusion test model 50
Figure 33 Flow chart of in vitro and ex vivo test 51
Figure 34 MG63 (magnification x10) 53
Figure 35 Push out test 55
Figure 36 Flow chart of in vivo test 56
Figure 37 The procedure of defects creation 58
Figure 38 The procedure of material injection 60
Figure 39 The XRD results of (a) CSD (b) CSH (c) CSH/HCL 66
Figure 40 Microstructure of (a) CSD powder and (b) αCSH powder 68
Figure 41 Thermal properties of (a) DSC and (b) TGA curve 70
Figure 42 The microstructure of (a) CSH/DD water and (b) CSH/HCl (magnification x250, x3000) 72
Figure 43Extrusion force of materials 73
Figure 44 pH value of CSH/DD water and CSH/HCl at 1, 7, 14, 21, 28, 35, 42, and 49 days 75
Figure 45 Weight loss of materials at 1, 7, 14, 21, 28, 35, 42, and 49 days. 76
Figure 46 Microstructure (magnification x1000, x3000) and the EDS analysis of CSH/DD water 78
Figure 47 Microstructure (magnification x1000, x3000) and the EDS analysis of CSH/HCl water 79
Figure 48 The MTS assay of materials 81
Figure 49 Cell morphology of CSH (magnification x10) (a) day 1 (b) day 3 (c) day 5 and (d) day 7 82
Figure 50 Cell morphology of CSH/HCl (magnification x10) (a) day 1 (b) day 3 (c) day 5 and (d) day 7 82
Figure 51 Cell morphology of Alginate (magnification x10) (a) day 1 (b) day 3 (c) day 5 and (d) day 7 83
Figure 52 The SEM morphology of CSH/DD water group (magnification x250, x1000) (a) day1 (b) day3 84
Figure 53 The SEM morphology of CSH/DD water group (magnification x250, x1000) (a) day5 (b) center of the scaffold at day5 85
Figure 54 The SEM morphology of CSH/HCl group (magnification x250, x1000) (a) day1 (b) day3 86
Figure 55 The SEM morphology of CSH/HCl group (magnification x250, x1000) (a) day5 (b) center of the scaffold at day5 87
Figure 56 At 4 weeks, CT results of 2D images (a) Material group (b) Defect group (NB= New Bone, M= Material, D= Defect) 90
Figure 57 The CT analysis of 2D images at 12 weeks, CT results of 2D images (a) Material group (b) Defect group ( NB=New Bone) 91
Figure 58 The CT analysis of 2D image of control group (*= Location of Hill-Sachs defect) 91
Figure 59 Results of Bone Volume/Tissue Volume 92
Figure 60 Results of 3D reorganization 94
Figure 61 Defect image of HE stain at 4 weeks (magnification x4, x10) (*= Location of Defect) (A=bottom of defect, B=center of defect, C=edge of defect) 96
Figure 62 Defect+ Material image of HE stain at 4 weeks (magnification x4, x10) (A=bottom of defect, B=center of defect, C=edge of defect) 97
Figure 63 Defect image of HE stain at 12 weeks (magnification x4, x10) (A=bottom of defect, B=center of defect, C=edge of defect) 98
Figure 64 Defect+ Material image of HE stain at 12 weeks (magnification x4, x10) (A=bottom of defect, B=center of defect, C=edge of defect) 99
Figure 65 Normal control of HE stain (magnification x4, x10) (W= woven structure, M= Material, NB= Newly Bone, CT= Connective Tissue. *= Location of Defect) 100
Figure 66 Defect image of Masson’s Trichrome stain at 4 weeks (magnification x4, x10) (*= Location of Defect) 102
Figure 67 Defect+ Material image of Masson’s Trichrome stain at 4 weeks (magnification x4, x10) 103
Figure 68 Defect +Material image of Masson’s Trichrome stain at 12 weeks (magnification x4, x10) 104
Figure 69 Defect image of Masson’s Trichrome stain at 12 weeks (magnification x4, x10) 105
Figure 70 Normal control image of Masson’s Trichrome stain (magnification x4, x10) (*= Location of Defect) 106
Figure 71 Injection of CSH/HCl/Alginate into bone defect diagram 113
Figure 72 Mechanism of bone remodeling by material degradation 115
Figure 73 Observation of the Hill-Sachs defect repaired by CSH/HCl/alginate. (a) 4 weeks (b) 12 weeks 120


List of Tables
Table 1 The suggestions of treatment for Hill-Sachs lesion (Armitage, et al., 2010; Lopez-Hualda, et al., 2014) 13
Table 2 Mechanical properties of nature bone and some ceramics and glass-ceramics materials 22
Table 3 Properties ofα and β phase (Singh ＆ Middendorf, 2007) 27
Table 4 The species of hydrogels(Nguyen ＆ Lee, 2010) 31
Table 5 Drugs of Experiment 36
Table 6 Samples of the materials 40
Table 7 Reagents of HE stain 62
Table 8 Reagents of Masson’s Trichrome stain 63
Table 9 Elements of quantitative 69
Table 10 Time of hardening reaction 71
Table 11 Viscosity of alginate concentration 74
Table 12 Appearance of samples 77

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