研發透明質酸攜帶牙髓幹細胞之磷酸鈣/硫酸鈣雙相水泥應用於植體周圍骨再生治療

Abstract

使用植體作為缺牙骨脊的重建治療選擇，在臨床上已經相當普遍。但拔牙後的缺牙骨脊多有缺損或後續吸收的情形。植體置入時若面臨周圍大範圍骨缺損，除了初期穩定度可能受影響外，植體的長期成功率及是否能避免後續植體周圍炎的發生，都需要倚賴有效的植體周圍骨再生治療。 傳統處理植體周圍骨再生的方式為利用再生隔絕膜和骨材來達成引導骨再生。但可吸收再生膜常有維持空間能力不佳的問題，常需要加上骨釘來撐起空間，不但操作較困難，對於大範圍缺損成功率也較低，因此開發此類材料，尚須補強再生金三角中的細胞和生長因子，以期能更有功效。磷酸鈣/硫酸鈣雙相骨水泥可提供骨再生所需材料及可塑型的特性。而牙髓幹細胞被證實具有容易取得及良好的增殖率的特性，對於骨再生中需要增加前驅細胞量的目標，是不錯的選擇。因此，我們選擇對幹細胞親和度高的透明質酸來攜帶牙髓幹細胞，並和磷酸鈣/硫酸鈣雙相骨水泥混合為複合材料應用於植體周圍骨再生。本實驗的目的主要在研發透明質酸攜帶牙髓幹細胞之磷酸鈣/硫酸鈣雙相水泥應用於植體周圍骨缺損重建。 本實驗第一部分先分離培養齒源幹細胞，並確認牙髓幹細胞相對最優的效能。接著第二、三部份利用生物相容性及物化性質各項測試確認材料的最佳化。第四部分利用最佳化材料進行免疫缺乏小白鼠(SCID mouse)的實驗，初步觀察牙髓幹細胞和條件培養液加入材料的效果良好，材料放置在小鼠頂骨的傷口癒合良好，牙髓幹細胞和條件培養液對於前3周炎性反應和降解礦化情形相較於控制組都有促進的發現。 最後第五部分進行大動物-犬隻的功效性實驗，分成 3 組，實驗組一，使用磷酸鈣/硫酸鈣雙相骨水泥加上2%透明質酸及條件培養液攜帶犬牙髓幹細胞(FC)；實驗組二，使用磷酸鈣/硫酸鈣雙相骨水泥加上2%透明質酸及條件培養液(F)；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、斷層掃描分析與組織切片判讀和骨頭螢光標定觀察等分析，評估人工植體周圍的骨再生效果。 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；在植體穩定度上，皆有達到臨床上可以接受的穩定度。在組織切片中計算的硬組織的植體覆蓋率(%)，八周及十二周組，兩實驗組F組(82±0.35,83.75±15.91)和FC組(81.25±5.30, 88.75±12.37)相較於控制組(38.35±13.31, 41.68±17.41)的覆蓋率都有達到統計上的差異，而在F組不同周間，其覆蓋率亦有達到統計上的差異。而在放射線影像及斷層掃描中分別計算的硬組織的植體覆蓋率，各組在各時段雖然都沒有達到統計上的差異，但仍可發現兩實驗組有高於控制組的趨勢。在螢光標定觀察下，兩實驗組的骨生成的時序性類似並相較控制組有促進效果；而在未脫鈣的組織切片中，我們亦可觀察到該時序性帶來的成骨差異。 加入牙髓幹細胞及條件培養液的磷酸鈣/硫酸鈣雙相骨水泥複合材料，有良好的生物相容性和適合骨再生治療的物化性質，動物實驗上材料應用於植體周圍骨損的再生手術，在組織學分析上植體覆蓋率的表現相較控制組有顯著差異，雖然放射影像分析未達顯著差異，但根據螢光標定及組織切片觀察上仍可由骨生成時序看到加入牙髓幹細胞及條件培養液的可能促進效果。透明質酸-條件培養液攜帶或不攜帶牙髓幹細胞之磷酸鈣/硫酸鈣雙相水泥應為植體周圍骨再生治療具潛力的選項。

It is common to have dental implant in edentulous ridge reconstruction. After the tooth extraction, the following ridge resorption and deficiency is big problem to long-term implant success rate and it may cause the peri-implantitis. Therefore, to develop an efficient biomaterial for peri-implant bone regeneration is important to implant dentistry. The most popular way to perform the guided bone regeneration is to apply a membrane as barrier and bone substitute as scaffold. It is often technique sensitive to reconstruct a large bony defect, but the clinicians may encounter the difficulty in space making. For better result, the development of biomaterial should consider more about the source of cells and growth factors for peri-implant bone regeneration. Calcium phosphate and calcium sulfate biphasic cements may provide the potential of hydroxyapatite deposition and moldable property during regeneration procedure. The dental pulp stem cells are proved to be a good candidate for increasing bone progenitors. In this study, the dental pulp stem cells were incorporated in hyaluronic acid to mix with calcium phosphate and calcium sulfate biphasic cements as scaffold for peri-implant bone regeneration. The study invetigate the superiority of dental pulp stem cell in proliferation and then try to figure out the best formula for this composite biomaterial by biocompatibility test and physicochemical test. The best formula was applied and operated in the calvarium model of SCID mouse. The performance of the biomaterial in wound healing was good, and it showed faster inflammatory response and mineralization in the group CPC/CSC mixed with a HA-conditioned medium hydrogel with/without dDPSC. In the following, three beagle dogs, implant placement and peri-implant bone regeneration were performed 3 months after tooth extraction in the mandible. Two standardized box-shaped defects were bilaterally created, and dental implants were placed in the center of the defects with a dehiscence of 4 mm. Three treatment modalities were randomly applied: i) CPC/CSC mixed with a dDPSC HA hydrogel with conditioned medium: ii) CPC/CSC mixed with a dDPSC HA hydrogel: iii) no bone augmentation (empty). After a healing period of 4 and 8 and 16 weeks, micro-CT and histological analyses were performed. The survival rate is 100% for all the implants but the implants were also with mucosal inflammation during observation period. Histomorphometric analysis revealed a greater marginal bone cover ratio(%) for groups in CPC/CSC mixed with a HA-conditioned medium hydrogel with/without dDPSC (82±0.35,83.75±15.91 at 8 weeks, 81.25±5.30, 88.75±12.37 at 16 weeks) compared to empty control (38.35±13.31 at 8 weeks, , 41.68±17.41 at 16 weeks). Regarding the regeneration process, CPC/CSC mixed with a HA-conditioned medium hydrogel with/without dDPSC were found to get superior healing potential at 4 weeks under fluorescence microscope observation of non-decalcified specimen. CPC/CSC mixed with a HA-conditioned medium hydrogel with/without dDPSC is a potential biomaterial for peri-implant bone regeneration.