第五章 ：結果與討論
5.3 體外生物活性
5.3.1 Alizarin Red S 定性染色
圖 5-24 和 5-25 分別為牙髓細胞與不同材料萃取液培養 14 天及 21 天後 alizarin red S 之定性染色結果。在顯微鏡下可觀察到在 14 天時有加入 100 & 400 ng/ml TGF-β1 的磷酸鈣-水膠材料組已開始有少量鈣結節(calcification nodule)之生 成，而對照組、CPC、MTA 則無觀察到鈣化生成。各組間在 21 天鈣結節差異更加 明顯，可觀察到在加入 100 & 400 ng/ml TGF-β1 的磷酸鈣-水膠材料組有大量的鈣 化生成，CPC、CPH 也有些許生成，MTA 和對照組則幾乎沒有。
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(A) (B)
(C) (D)
(E) (F)
圖 5-24 牙髓細胞十四天 Alizarin red S 定性染色 (A) Negative control; (B) MTA;
(C) CPC; (D) CPC/ hydrogel; (E) CPC/ hydrogel/ 100 ng/ml TGF-β1; (F) CPC/
hydrogel/ 400 ng/ml TGF-β1
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(A) (B)
(C) (D)
(E) (F)
圖 5-25 牙髓細胞二十一天 Alizarin red S 定性染色 (A) Negative control; (B) MTA;
(C) CPC; (D) CPC/ hydrogel; (E) CPC/ hydrogel/ 100 ng/ml TGF-β1; (F) CPC/hydrogel/ 400 ng/ml TGF-β1
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5.3.2 Alizarin Red S 定量染色
圖 5-26 和 5-27 分別為牙髓細胞與不同材料萃取液培養 14 天及 21 天後 alizarin red S 之定量測試結果。在第 14 天時，所有磷酸鈣材料組的鈣生成量皆顯 著高於 MTA 及對照組。而這個差距在 21 天更加拉大：CPC/ hydrogel/ 400 ng/ml TGF-β1 鈣化生成量顯著的高過其他所有組別，CPC/ hydrogel/ 100 ng/ml TGF-β1、
CPC/ hydrogel/ 400 ng/ml TGF-β1、及 CPH 則是顯著高於 CPC、MTA、及對照組。
綜合體外生物活性的測試結果，alizarin red S 定性的實驗結果顯示，加入 TGF-β1 磷酸鈣材料組比其他材料更早出現顯微鏡下可見之鈣結節 (calcification
因此動物實驗採用 CPC/ hydrogel/ 400 ng/ml TGF-β1 材料與 CPC/ hydrogel，評估加 入 TGF--β1 於活體的療效，並採用 MTA 作為對照組。
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圖 5-26 牙髓細胞十四天 Alizarin red S 定量測試
圖 5-27 牙髓細胞二十一天 Alizarin red S 定量測試
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圖 5-29 CPC/ hydrogel/ 400 ng/ml TGF-β1 之動物實驗兩個月 μ-CT 影像。(C: 於 實驗中放置之 CPC。箭頭 ：牙本質橋生成。)
圖 5-30 MTA 之動物實驗兩個月 μ-CT 影像。(M: 於實驗中放置之 MTA。箭頭 ： 牙本質橋生成。箭號 ：不連續的牙本質橋)
C
C
M
M
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表 5-1 μ-CT 影像硬組織生成評估
Hard tissue formation Continuity of dentin bridge CPH/400ng/mL TGF-β1 6/6 (100%) 6/6 (100%)
CPH 6/6 (100%) 4/6(67%)
MTA 2/2(100%) 1/2(50%)
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第七章：未來研究方向
1.
以組織切片分析動物實驗之結果：細胞發炎程度、牙本質橋生成型態及是否有 缺陷、是否有微生物感染，進一步探討本材料對於牙髓再生/修復的效果。2.
設計更長測試時間、或同一個體連續時間測試的動物實驗，以觀察材料的降解 是否伴隨細胞長入及取代材料空間。3.
分析磷酸鈣材料再結晶相轉換的過程是否有利於鈣離子釋放，以及探討離子釋 放對於細胞生成鈣化結節或牙本質橋之影響。77
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