根管壁磷酸鈣沈積於誘導牙本質再生之應用

Abstract

傳統人工合成的氫氧基磷灰石(hydroxyapatite, HAP）為結晶性良好的磷酸鹽材料，是臨床醫療常見的人工骨粉材料，在做為骨傳導（osteoconduction）的應用上具有相當良好的療效。近年研究發現生物體中的HAP隨著硬組織的不同而呈現不同的次級結構，在生物礦化過程扮演重要調控角色。因此開發HAP前驅物或晶體結構較不穩定之磷酸鈣材料，用於生物硬組織再生研究成為新興的課題。由於感染的根管在進行情創與修形後，會影響根管表面硬組織的形態特徵與組成性質，在後續若要進行牙本質-牙髓再生（dentin-pulp regeneration）處置時可能不利於幹細胞的貼附、生長與分化。因此本研究目的是利用沈積法在根管內表面形成一層均質覆蓋且結晶性不佳的磷酸鈣沈積物，透過可釋放鈣離與磷酸根離子的特性吸引並調控幹細胞的生物礦化行為，並且改變根管表面形態特徵以利於幹細胞貼附生長。本研究首先使用接近飽和濃度的10％ Na2HPO4與不同濃度的CaCl2溶液進行沉澱反應來製備磷酸鈣沈積物，從產物結晶尺寸與反應酸鹼值結果，選擇0.05 M與0.1 M CaCl2進行後續實驗。接著在牙本質樣本的根管表面直接進行沈積反應，評估反應時間、CaCl2濃度對於牙本質表面之磷酸鈣沈積物形態、組成成分、晶體結構與覆蓋牙本質表面行為的影響效應。最後再以牙根根管模型進行研究，測試溶液沖洗位置與CaCl2濃度對於根管表面形成磷酸鈣沈積物的覆蓋行為。 掃描式電子顯微鏡(scanning electron microscope, SEM)分析發現，當反應時間大於15分鐘時，無論是0.05 M與0.1 M CaCl2組均可於牙本質表面形成高覆蓋率的磷酸鈣沈積物，主要呈現表面針刺狀或條狀結晶覆蓋的球狀結構，與HAP結構相似，0.1 M組在反應時間大於30分鐘時開始可觀察到少量片狀結晶散布於球狀主結構中。此外隨著反應時間與CaCl2濃度增加，穿透式電子顯微鏡（transmission electron microscope, TEM）分析發現產物鈣磷比從1.31增加到1.47，同時產物晶體也逐漸呈現方向性排列形態，而選區電子衍射（selected area electron diffraction, SAED）的晶體繞射點亦趨明顯，顯示產物結晶性提升，其結構鑑定為缺鈣型氫氧基磷灰石（calcium-deficient hydroxyapatite , CDHA）。覆蓋率分析方面，當反應時間為15分鐘時，0.05 M組平均覆蓋率為97.82%、平均覆蓋厚度為18.71 µm，0.1 M 組平均覆蓋率為97.57%、平均覆蓋厚度為12.79 µm，兩組的平均覆蓋率與覆蓋厚度相似，沒有統計顯著差異。於牙根根管模型的研究結果發現，針頭置於根管中段進行沖洗所形成磷酸鈣沉積物覆蓋情況最佳，當反應時間為15分鐘時，0.05 M組的產物均呈現球狀結構，其平均覆蓋率為93.39 %，0.1 M 組則除了球狀結構外亦有片狀結構出現，其平均覆蓋率為95.88 %，兩者間沒有統計顯著差異。 總結來說，將針頭置於根管中段沖洗0.05 M CaCl2 15分鐘，可在牙根根管樣本表面形成覆蓋率高、均值度佳、低結晶性的磷酸鈣沈積物，推測將有利於調控齒源性幹細胞的生物礦化行為，進而達到促進牙本質與牙髓組織再生的結果。

Artificial hydroxyapatite(HAP) is a kind of calcium phosphate with well crystallized and good biocompatibility. It is widely used in bone graft material because good osteoconductive ability results in good treatment outcomes. There are several HAP substructures in different hard tissues, and it plays an essential regulatory role in the biomineralization process. Therefore, developing HAP precursors or calcium phosphate materials with relatively unstable crystal structures for studying biological hard tissue regeneration has become an emerging topic. The morphological characteristics and composition of the hard tissue on the surface of the root canal might be affected by canal debridement and enlargement, and may be unfavorable for subsequent dentin-pulp regeneration for attachment, growth, and differentiation of stem cells. Therefore, the purpose of this study is to use the precipitation method to form a layer of calcium phosphate deposits with homogeneous coverage and poor crystallinity on the inner surface of the root canal. Change the morphological characteristics of root canal surface to facilitate the growth of stem cells. In this study, we use the precipitation method to form calcium phosphate using a serial application of a 10% Na2HPO4 and different concentrations of CaCl2 and observe the appropriate crystal form and size under the optical microscope as a result of an ionic reaction. Considering the neutral pH value and small crystal size of calcium phosphate formation, we choose 0.05M and 0.1M CaCl2 as ideal concentrations and apply them to the following experiment. Then, the deposition reaction was performed directly on the dentin samples. The effect of reaction time and CaCl2 concentration on the morphology, composition, crystal structure, and behavior of covering the dentin surface of calcium phosphate deposits were evaluated. Finally, the root canal samples were used to study the coverage behavior by different solution irrigation positions and CaCl2 concentration in forming calcium phosphate deposits on the root canal surface.