新型鈣矽類化合物之抗菌性、基因毒性、發炎效應與骨頭誘導生成之探討

The Investigation of Calcium Silicate Cements on Antibacterial effect, Genotoxicity, Inflammatory Reaction and Osteo-conduction

陳志麟(Chih-Lin Chen)

指導教授 ： 高嘉澤 ； 黃翠賢

中山醫學大學/口腔醫學院/口腔生物暨材料科學研究所/博士(2011年)

https://doi.org/10.6834/CSMU.2011.00064

摘要

當根管治療過之牙齒發生根尖周圍炎，有可能須接受根尖切除
手術，術中須將根尖位置以逆充填之材料填補，以達修復之作用。常
用之根尖充填材料如銀粉、氧化鋅丁香油水門酊、氫氧化鈣、環氧樹
脂材料和鈣矽水門酊（如三氧礦化合物MTA)等。良好的根尖充填材
料要具備有穩定的物理化學性質，如良好的封閉性、不溶於水、與牙
齒有好的黏著力、Ｘ光不透射等。更要具有良好的生物相容性，如不
具細胞毒性、基因毒性等。目前常用之根尖充填劑- 三氧礦化合物
MTA，優點除可用於根尖充填外，另外可以作牙髓穿孔修補，覆髓。
其缺點為硬化時間過長與費用過高。
本實驗室過去曾已自行開發出相似於鈣矽類充填材料（CS），過
去研究證明其物理與化學性質與商品之MTA 相似，包括其硬度、酸
鹼值、硬化時間與水合後之結晶結構。但是本材料CS 對於生物學上
之效應，仍未有足夠證據顯示其優缺點。
因此本研究目的如下：
一、比較評估本材料CS 與其它根尖充填材料對於細菌之抗菌性差異。
二、探討本材料CS 對於細胞之基因毒性。
三、比較本材料CS 與MTA 根尖充填劑對於細胞發炎效應。
四、探討 CS 與MTA 對於骨細胞之作用。
研究材料與方法
所有測試之材料，商品化部份均依照廠商指示混合，自製的CS 材料
依水粉比0.3cc: 1gram 比例混合。
一、抗菌性試驗
材料使用銀粉、氧化鋅丁香油水門酊、MTA 和CS。採用agar diffusion test 和alamar blue assay ， 以 Streptococcus sanguinis, Streptococcus mutans 和 Escherichia coli.作為檢測之細菌。結果以one way ANOVA 統計分析結果。
二、細胞基因毒性分析
首先以粒腺體呈色分析先測試MG63 細胞之細胞存活率。再以DNA 沈澱反應分析材料作用於細胞後之基因傷害程度。
三、細胞發炎測試
首先以粒腺體呈色分析先測試牙髓細胞之細胞存活率。利用免疫螢光反應，檢測細胞之 focal adhesion kinase (FAK) 和 COX-2 蛋白
質分佈。利用RT-PCR 分析COX-2 基因之表現。
四、骨頭生成反應分析
以 X-ray diffraction (XRD) 分析材料之水合後結晶結構。利用西
方墨點分析ERK kinase 表現。以RT-PCR 檢測骨細胞生成因子表現。
結果
在agar diffusion test 中，只於IRM 這組與S. sanguinis 和 E. coli agar plates 有出現抑制環(inhibition zone)。 於Alamar blue 分析，觀察於1, 3, 6, 12 hr 小時中，也是於S. sanguinis 和 E. coli agar plates有出現低的呈色反應。 而S. mutans 此株細菌對於所有材料皆無被抑制之反應出現。
基因毒性試驗中，MG63 對於MTA 或CS 材料均具細胞相容性，
且對於細胞不具有基因毒性。
發炎效應檢測上發現：MTA 和CS 材料，對於人類牙髓細胞具有
生物相容性；FAK 蛋白明顯表現於培養基上；二種材料同時會對細
胞產生COX-2 表現之炎性反應，但隨時間增加而減低炎性反應。
骨細胞之骨生成測試發現， MTA 和CS 材料作用於骨細胞株(MG63)後，p-ERK kinase 訊息蛋白表現增強。對於骨生成相關因子：
type I collagen (Col), osteocalcin (OC), alkaline phosphatase (ALP), Bone sialoprotein (BSP) 和 osteopontin (OPN)蛋白之表現均明顯增加。
結語
由以上試驗發現，CS 與MTA 材料對於S. mutans, S. sanguinis 和
E. coli. 三株細菌不具有抗菌性；對於骨細胞也不具有基因毒性；
牙髓細胞接觸後，會有發炎反應，隨時間增加而炎性減弱；對於骨細
胞具有刺激骨傳導(osteoconduction)之反應。

關鍵字

新型鈣矽填充材料 ； 三氧礦化物 ； 生物相容性 ； 發炎反應 ； 基因

並列摘要

The periapical inflammation may be appeared post root canal therapy. The tooth apicoectomy will be performed. After root apex was dissection, a lot of materials were used to seal apical foramen. They were amalgam, zinc oxide eugenol, super epoxi base resin, calcium hydroxide or calcium silicate cement (mineral trioxide aggregate, MTA). The benefits of MTA were used not only at root apex but also can used on perforation of canal walls. The absence of MTA was too expensive and long setting time.
Our laboratory recently developed new calcium silicate cement (CS), with similar physical and chemical characteristics of MTA. The setting time was improved, pH was alkaline range, acceptable hardness and hydration compound. It is lack of biologic effects of this new CS
material.
The purpose of present study were
1. to evaluate the antibacterial activity of leachable components of root-end filling materials (IRM, amalgam, MTA and CS) against three different types of microorganisms utilizing both the ADT and AB assays, in vitro study.
2. to investigate the genotoxicity of cells cultured with MTA and CS cements by the DNA precipitation assay.
3. to investigate the expression of the inflammatory marker COX-2 in primary culture dental pulp cells cultured with MTA or CS cements.
4. to compare the biocompatibility of both MTA and the newly
developed CS cement, and to evaluate their bone marker
expression in bone cells.

Materials and Methods

1. Antibacterial Effects Assay
The root-end filling materials were mixed according to the
manufacturers' directions and then placed into 6-mm diameter Teflon rings with mixed materials; they were set for 24 hours, then tested.
Antibacterial activity of the root-end filling materials was evaluated utilizing Streptococcus sanguinis, Streptococcus mutans and Escherichia coli. The antibacterial activity of the root-end filling materials tested was determined by measuring the diameter of each of the zones of inhibition (agar diffusion test). The Alamar Blue assay was used to detect bacterial growth. Statistical analysis was conducted using one-way analysis of variance.
2. Genotoxicity Assay
A mitochondrial colorimetric assay was used to evaluate the MG63 survival rate. A DNA precipitation assay was used to detect the MG63 DNA damage after contact with MTA or CS cement extracts.
3. Inflammation Assay
The mitochondrial colorimetric assay was used to evaluate pulp cell survival rates. Fluorescent immunohistochemistry was used to observe focal adhesion kinase (FAK) and COX-2 distributions in the cells. RT-PCR was used to assess COX-2 expression.
4. Osteo Formation Effect Assay
X-ray diffraction (XRD) was employed to identify material surface structure, and tetrazolium bromide colorimetric assay (MTT) was used to evaluate the cell viability. The relative mitogen activation protein kinase expression was compared with Western blot, and bone marker expression
was evaluated with reverse transcriptase polymerization chain reaction (RT-PCR).

Results

The result showed zones of inhibition were observed in the IRM group treated with S. sanguinis and E. coli agar plates in an agar diffusion test. The cultures of S. sanguinis and E. coli showed the lowest absorbance
with the IRM group at different time intervals of observation (1, 3, 6, 12 hr) (p < 0.05). The growth of S. mutans showed no statistical difference between controls and all tested materials (p > 0.05).
The results showed that MTA and CS are cytocompatible with MG63 cells. There was no significant difference in the survival rate with MTA and CS materials (p> .05). Neither MTA nor CS cements causing MG63 cell DNA damage showed significant genotoxicity (p>.05).
The results showed that MTA and CS are biocompatible with pulp cells (p>0.05). FAK was well distributed in pulp cells in contact with both cements. Both MTA and CS cements induced pulp cell inflammation as evidenced by increased COX-2 expression The results showed that CS and MTA are similar chemical structures and biocompatible with MG63 cells. CS and MTA cements showed good MG63 cell proliferation by high phosphor extracellular signal-regulated kinase (p-ERK) expression levels. CS and MTA cements showed the evident type I collagen (Col), osteocalcin (OC), alkaline phosphatase (ALP), Bone sialoprotein (BSP) and osteopontin (OPN) expression.

Conclusions

It is concluded that both the agar diffusion test and the Alamar blue assay gave comparable findings assessing the antimicrobial activity present in root-end filling materials. No antimicrobial activity was detected for MTA, CS and amalgam after coming into contact with S. mutans, S. sanguinis and E. coli. IRM showed high antimicrobial activity with both S. sanguinis and E. coli. Both MTA and CS cements are compatible with MG63 cells, and they are not cancer causing agents. Both MTA and CS cements induced pulp cell inflammation as evidenced by increased COX-2 expression. Both MTA and CS cements are
biocompatible and appear to have osteoconduction effects on bone cells.

並列關鍵字

Calcium silicate cement ； MTA ； antibacterial effect ； inflammation ； western blot assay ； RT-PCR ； genotoxicity ； osteoconduction

參考文獻

Chen CL, Kao CT, Ding SJ, Shie MY, Huang TH. Expression of

aggregate (MTA) preparations against Enterococcus faecalis and Streptococcus sanguis in vitro. J Endod 2006;32: 1053–6.

Anso N, Hoahino E. Predominant obligate anacrohes invading the deep layers of root canal dentine. Int Endodon J 1990;23:20-7.

Asgary S, Parirokh M, Egbbal MJ, Brink F. Chemical differences between white and gray mineral trioxide aggregate. J Endod 2005;31:101–3.

Aubin JE. Bone stem cells. J Cell Biochem Suppl 1998;30–31: