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研究生:蔡尚節
研究生(外文):Shang-Jye Tsai
論文名稱:研發新型磷酸鈣複合式骨材合併齒源性幹細胞應用於齒槽骨再生
論文名稱(外文):Development of Novel Calcium Phosphate Composite Bone Cement in Application for Alveolar Bone Regeneration with Odontogenic Stem Cell
指導教授:林俊彬林俊彬引用關係章浩宏章浩宏引用關係
口試委員:林江珍李志偉廖運炫林弘萍
口試日期:2018-06-02
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
論文頁數:128
中文關鍵詞:新型磷酸鈣複合式骨材牙髓幹細胞透明質酸明膠磷酸氫二鈉
外文關鍵詞:Calcium phosphate and calcium sulfate cementsodontogenic stem cellGelatinhyaluronic acidperi-implant regeneration
DOI:10.6342/NTU201802601
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骨質缺損在牙科領域內一直是不易克服的難題,若以高純化第一型膠原蛋白充填拔牙後窩洞,可以獲得不錯的效果,但是如果遇到更加困難的骨缺損,如嚴重骨缺損(critical bone defect) ,則可能需要自體骨移植才能有不錯的效果,缺點是病患可能因為需要接受多處而複雜的手術,徒增手術不適,在講究較小侵入性手術(minimal invasive surgical procedure)的趨勢下,研發出較佳的促進骨癒合的材料,並能展現可預期的骨再生效果,對臨床極為重要。

本研究之目的在研發一種應用於齒槽骨缺損且具適合齒源性幹細胞生長及加快降解速率反應之骨水泥,擬合併磷酸鈣(Calcium phosphate cement, CPC)及硫酸鈣的骨水泥(calcium sulfate cement, CSC)來調製,本實驗將由調配出一具有降解快速,生物親和力佳的類HAp結晶型態加速生成新式磷酸鈣骨水泥(FS+CS)的開始,漸次加入具有增加磷酸鈣骨水泥結晶比例的明膠(Gelatin, G)、具有增強細胞親合度佳的透明質酸透明質酸(Hyaluronic acid, HA)及過飽和的磷酸氫二鈉(Disodium Hydrogen Phosphate,DHP)水溶液而形成最佳化的調拌配方。

為達本研究目的,研究計分五部分逐步完成,第一部份先確立人類牙髓齒源性幹細胞於不同透明質酸下的最佳生長濃度;第二部分由生物相容性及物理化學性質確定配方,結果並顯示加入磷酸氫二鈉(Disodium Hydrogen Phosphate)有助於降解速率提升, 0.1g CS這組的降解速率於第1天即可達到64.24%相較於市售骨材CPC第1天即可達到97.92%,有統計上顯著的差異。而加入氫氧化鈉(NaOH)有助於降解速率提升,0.1 g CS這組的降解速率於第4天即可達到74.21%相較於市售骨材CPC第4天即可達到97.23%,有統計上顯著的差異。第三部分藉由大鼠頭蓋骨骨膜下注射模型驗證所研發之配方具有誘導骨生長能力,結果顯示實驗配方組別(FS+CS+HA+NaOH)組及(FS+CS+HA+G+DHP)組具有骨礦化程度高並且炎性反應較低。第四部分在小動物的實驗結果下,進一步以免疫組織化學(IHC)染色確認配方骨水泥具有誘導成骨細胞(osteoblast)含有Runx2轉錄因子表現的證據。並選擇骨誘導能力較佳之組別(FS+CS+HA+G+DHP)組進行大動物試驗評估。第五部分進行大動物功效性實驗,於下顎目標牙齒拔後3個月後於雙側創造4微米深的長方形植體旁骨缺損後,分成 4 組,實驗組一,使用CPC/CSC加上透明質酸及明膠及磷酸氫二鈉(Disodium Hydrogen Phosphate) DHP水溶液,培養液攜帶犬牙髓幹細胞(FS+CS+G+HA+DHP+Cell);實驗組二,使用CPC/CSC加上透明質酸及明膠及磷酸氫二鈉(FS+CS+G+HA+DHP);實驗組三,使用CPC(FS);控制組不使用材料為empty空白組(Con)。分別於植入後第 4 週、第 8 週與第 12 週 進行植體穩定度分析、放射線影像分析、斷層掃描分析,評估牙科植體周圍的骨缺損的骨再生效果。大動物的犬隻實驗結果,各組植體的存活率是100%,但仍伴隨程度不一的植體周圍炎;在植體穩定度(periotest)上,皆有達到臨床上可以接受的穩定度,且(FS+CS+HA+G+DHP+Cell)組相較於(FS+CS+HA+G+DHP)組於十二週組比較時出現統計上顯著差異,帶有幹細胞的組別的穩定度(periotest)改善率在近遠心及頰舌側值(33.3;23.6)相較於未攜帶幹細胞的組別的穩定度(periotest)改善率值(5.9;1.3)。
本研究研發出一種合併牙髓幹細胞之新型磷酸鈣複合式骨材,其對於植體周圍嚴重骨缺損,可以提供極具潛力且可預期之骨再生。
Bone defect repair in dental clinical practice is always a difficult task for oral and maxillofacial surgeon. Pure type-1 collagen filling post-extraction socket could promote wound healing and bone density gain. However, when repairing critical bony defect within oral cavity, autogenous bone graft method may be a good choice. But multiple site interventions are usually against the trend of minimal invasive surgical procedure. Therefore, new biomaterials with simplified and predictable outcoming is still in investigation.

The goal of this study was to develop a novel bone cement with fast degradation rate and suitable for odontogenic stem cells growth within alveolar bony defect. Calcium phosphate cement (CPC) and calcium sulfate cement(CSC)were used to develop a novel bone cement with fast degradation rate, good biocompatibility and crystallinity morphology approximate hydroxyapatite properties. By means of adding super saturation solution of disodium hydrogen phosphate, hyaluronic acid and gelatin in CPC/CSC to gain properties mentioned above. The study investigates the superiority of odontogenic stem cell in proliferation of hyaluronic acid concentration and then try to determine the best formula for this composite biomaterial by biocompatibility test and physicochemical test. The results revealed that disodium hydrogen phosphate(DHP) solution promote formula degradation rate (64.24%,1st day) while comparing to CPC (97.92%,1st day) with statistically significant. Furthermore, sodium hydroxide(NaOH) solution promote formula degradation rate (74.21%,4th day) while comparing to CPC (97.23%,4th day) with statistically significant.
Formula were then applied and operated in the calvarium model of Winstar rats. The osteoconduction performance of the biomaterial was good, and it showed less inflammatory response and better mineralization in the group of CPC/CSC mixed with hyaluronic acid, NaOH and CPC/CSC mixed with hyaluronic acid, gelatin and DHP.
Experiment groups were exam with Runx2 through immune histology chemistry staining to identify which biomaterial induce better osteoblast mineralization performance. Formula (CPC/CSC/HA/Gelatin/DHP) with good osteoconduction was chosen to carry on further functional assay on large animal model.
Simutanous critical bone defect created around implant and repaired with test material were perfomed 3months later following tooth extraction in four beagle dogs Two standardized box-shaped critical bone defects were created bilaterally, and dental implants were placed in the center of the defects with a dehiscence of 4 mm. Four treatment modalities were randomly applied: i) CPC/CSC mixed with hyaluronic acid, gelatin, DHP solution and also with dDPSC: ii) CPC/CSC mixed with a hyaluronic acid, gelatin, DHP solution but without dDPSC: iii) CPC mixed with ddH2O: iv) no bone augmentation (empty). After a healing period of 4 and 8 and 12 weeks, micro-CT, x ray, and perio-test value analyses were performed.
The survival rate is 100% for all the implants but the implants were also with mucosal inflammation during observation period. Periotest value analysis revealed a greater improvement value for groups in CPC/CSC mixed with hyaluronic acid, gelatin and DHP with dDPSC (33.3 at mesial and distal aspect;23.6 at buccal and lingual aspact) compared to CPC/CSC mixed with hyaluronic acid, gelatin and DHP without dDPSC (5.9 at mesial and distal aspect;1.3 at buccal and lingual aspact) in 12 weeks group observation with statistically significant.
We thus concluded that a new bone coment based on combination of CPC/CSC with hyaluronic acid, gelatin and DHP with dDPSC is a potential biomaterial for critical peri-implant bone regeneration.
致謝 I
中文摘要 III
ABSTRACT V
目錄 VII
圖目錄 XI
表目錄 XVIII
第一章 導論 1
第二章 研究背景與文獻回顧 2
2.1拔牙後施行齒槽骨脊保存術有助拔牙窩洞癒合現況 2
2.1.1膠原蛋白代謝吸收迅速,不易持久,就影像學分析而言似有放射不透性(radiopacity)增加的現象 3
2.1.2氫氧磷灰石(Hydroxyapatite)置入拔牙後窩洞促進齒槽骨質增生 3
2.2磷酸鈣骨水泥Calcium phosphate(CAP) cement 4
2.2.1 α相三鈣磷酸鹽 (α Tri-calcium Phosphate,α-TCP)的降解 4
2.2.2牙科用骨水泥硫酸鈣(Calcium Sulfate,CaSO4)及特性 5
2.2.3 α-TCP與二水硫酸鈣Calcium Sulfate Dihydrate ( CSD;CaSO4·2H2O )的關聯性 6
2.2.4 HAp的特性 7
2.2.5磷酸鈣骨水泥的分類 8
2.3改善骨水泥的方法 9
2.3.1透明質酸(Hyaluronic acid,HA)提供組織細胞親水性 9
2.3.2明膠(Gelatin)加速磷酸鈣骨水泥硬化時間及增進細胞貼附及結晶形成 9
2.3.3 磷酸氫二鈉精準改善骨水泥酸度過高及減少骨水泥setting time 14
2.3.4 二水硫酸鈣(Calcium Sulfate Dihydrate,CSD)改善α-TCP及氫氧磷灰石(Hydroxyapatite,HAp)骨水泥不易降解吸收的方法 15
2.4 Runx2轉錄因子 16
第三章 研究目的 17
第四章 材料與實驗方法 18
4.1人類牙髓齒源性幹細胞的特性 18
4.1.1 細胞計數 18
4.1.2 人類牙髓齒源性幹細胞及人類胚胎顎間質細胞(Human embryonic palatal mesenchymal cells)進行不同濃度透明質酸(Hyaluronic acid,HA)的細胞生長活性測試 20
4.2調拌透明質酸-明膠混成磷酸鈣及硫酸鈣骨水泥(Hyaluronic acid-Gelatin hybridized Calcium phosphate and Calcium sulfate bone cement)方法 20
4.2.1 FS 的製備 21
4.2.2 CS的製備 21
4.2.3 HA的製備 21
4.2.4 Gelatin水溶液的製備 22
4.2.5 Na2HPO4水溶液的製備 22
4.2.6 NaOH水溶液的製備 22
4.3細胞活性及細胞毒性測試 23
4.4細胞直接貼附實驗 24
4.4.1 免疫螢光染色 24
4.4.2 PKH26 染色 24
4.5材料物化性質分析 25
4.5.1掃描式電子顯微鏡- SEM 25
4.5.2 表面元素分析-掃描式電子顯微鏡-能量散佈分析儀(SEM-EDS) 25
4.5.3 酸鹼值評估(pH Variation) 26
4.5.4 硬化時間(Setting Time) 26
4.5.5 抗壓強度測試(Compressive Strength) 26
4.5.6 降解測試(Degradation test) 27
4.5.6.1 測試樣本製備 27
4.5.6.2 測試方法 27
4.5.7 X光繞射分析(X-ray Diffraction analysis) 27
4.5.8 傅立葉轉換紅外線光譜(FT-IR) 27
4.6 小動物實驗 28
4.6.1 實驗目標 28
4.6.2 實驗方法 28
4.6.3 實驗組別及過程 28
4.6.4 標本製作流程 31
4.6.5標本骨生成量量化流程 31
4.6.6以免疫組織化學(IHC)染色確認含有Runx2轉錄因子表現的證據 32
4.7 大動物實驗 33
4.7.1 實驗設計 33
4.7.2 實驗動物的麻醉 33
4.7.3動物實驗手術步驟 34
4.7.3.1第一階段的手術步驟 34
4.7.3.2第二階段手術前設計植體周圍骨塊 35
4.7.3.3第二階段的手術步驟 37
4.7.4 臨床觀察及照顧 39
4.7.5 植體穩定度測量 39
4.7.6 動物犧牲與標本取得 40
4.7.6.1 福馬林藥水的製備 40
4.7.6.2 動物的犧牲 40
4.7.6.3 標本的取得 41
4.7.7 放射線影像分析與斷層掃描 41
4.7.7.1 放射線影像分析 41
4.7.7.2 斷層掃描 42
4.7.8 標本製作 42
4.7.8.1 標本的初步切割 42
4.7.8.2 含植體磨片標本製備 42
4.7.9 骨頭螢光標定 43
4.7.9.1標定方法 43
4.7.9.2 螢光染劑 43
4.7.9.3 螢光顯微鏡觀察 44
4.7.9.4 植體周圍骨頭覆蓋率 44
4.7.9.5 統計方法 45
第五章 實驗結果與討論 46
5.1人類牙髓齒源性幹細胞的特性 46
5.1.1細胞計數及觀察 46
5.1.2 人類牙髓齒源性幹細胞及人類胚胎顎間質細胞(Human embryonic palatal mesenchymal cells)進行不同濃度透明質酸(Hyaluronic acid,HA)的細胞生長活性測試 47
5.2調拌Hyaluronic acid-Gelatin hybridized Calcium phosphate and Calcium sulfate cement方法 50
5.3 生物相容性評估 55
5.3.1 細胞存活率實驗(cell viability assay)和材料共培養倒立顯微鏡觀察 55
5.3.2 細胞存活率實驗(cell viability assay)細胞直接貼附實驗(cell attachment on material)和材料直接接觸生長以PKH26染色螢光顯微鏡觀察 55
5.4 材料物化性質分析(Physicochemical Properties) 56
5.4.1 SEM 56
5.4.2 SEM-EDS表面元素分析-掃描式電子顯微鏡-能量散佈分析儀(SEM-EDS) 60
5.4.3酸鹼值評估(pH Variation) 65
5.4.4 硬化時間(Setting Time) 66
5.4.5 抗壓強度測試(Compressive Strength) 67
5.4.6 降解測試 68
5.4.7 FTIR 70
5.4.8 XRD 71
5.5 小動物實驗 75
5.5.1 臨床外觀 75
5.5.2 放射影像X光照射及微電腦斷層Micro CT 78
5.5.2.1 放射影像X光照射 78
5.5.2.2電腦斷層切片 79
5.5.3 脫鈣片HE染色 85
5.5.4 Runx2 IHC 染色 91
5.6 大動物實驗 97
5.6.1 外觀觀察 97
5.6.2 含植體磨片標本製備 100
5.6.3 植體穩定度測量 103
5.6.4 斷層掃描與放射線影像分析 105
5.6.5 大動物下顎骨螢光標定 108
5.7 配方適合齒源性幹細胞生長的原因 111
5.7.1 配方的化學官能基與骨傳導間的關係 111
5.7.2 配方容易降解可被被蝕骨細胞吸收轉變成成骨細胞 115
5.7.3 配方的結晶過程有利於細胞貼附 117
第六章 總結 122
參考文獻 124
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