研發透明質酸攜帶膠原蛋白酶及Simvastatin之磷酸鈣/硫酸鈣雙相生醫材料應用於活髓治療

Abstract

牙髓為具有修復與再生能力之組織，在治療具可回復性的牙髓傷害時，藉由適當的覆髓材料，可刺激形成牙本質橋之屏障阻擋外界刺激並保護剩餘健康的牙髓以保留牙髓的活性。近年來，牙髓再生的組織工程研究漸漸受到重視，在部分移除牙髓甚至完全移除牙髓的情況下，利用合適的骨架材料攜帶藥物或生長因子，期望引導剩餘牙髓或根尖組織之幹細胞至缺損部位，達到牙髓/牙本質再生的目的。 本研究之目的為利用磷酸鈣/硫酸鈣雙相材料為骨架，並以透明質酸攜帶Simvastatin及膠原蛋白酶，以調拌性質、硬化時間、降解測試、電子顯微鏡觀察及能量散射光譜分析材料性質及水合結晶產物；並進行雙相材料攜帶Simvastatin及膠原蛋白酶之藥物釋放測試；此外，利用人類牙髓幹細胞進行體外生物相容性測試，包括以AlamarBlue測試細胞存活及LDH測試細胞毒性。最後以米格魯犬作為模型進行動物實驗，並以micro–computed tomography (μ-CT)影像及組織學切片觀察分析。 結果顯示磷酸鈣加入硫酸鈣後，可降低其硬化時間為15-20分鐘，符合臨床操作性質。此材料水合後會形成具有孔洞之表面結構，有利於細胞生長，Simvasttin與膠原蛋白酶在一小時內即有大量釋放之現象，一天內即可達到總釋放量之一半；AlamarBlue與LDH的結果顯示雙相材料無論是否加入Simvastatin或是膠原蛋白酶，對於人類牙髓幹細胞之生物相容性良好；動物實驗中，含有Simvastatin及膠原蛋白酶的雙相材料可誘導牙本質的再生，並隨著材料之降解，鈣化組織會長入缺損之中。因此含Simvastatin及膠原蛋白酶之磷酸鈣/硫酸鈣雙相材料相當具有潛力作為活髓治療以及牙髓/牙本質再生的材料。

The dental pulp is able to repair and regenerate itself. To treat the reversible pulpitits, we can stimulate the formation of the barrier of the dentin bridge by blocking the external stimuli and protecting the remaining healthy pulp with appropriate capping material to maintain the pulp vitality. In recent years, tissue engineering in pulp regeneration has been paid more and more attention. In the case of partial removal of pulp or even complete removal of pulp, we hope to attract the stem cells from remaining pulp or apical tissue to the by use of appropriate scaffold to carry drugs or growth factors to achieve the purpose of pulp / dentin regeneration. The aim of this study was to evaluate the feasibility of biphasic calcium phosphate/calcium sulfate hemihydrates (CPC-CSH) biomaterial containing Simvastatin(Sim) and Collagenase(Col) for vital pulp therapy. Combination of CPC with various amounts of CSH was tested for handling property, setting time, degradation and SEM-EDS observation. Drug releasing pattern of Simvastatin and Collagenase combined with CPC-CSH was analyzed. In vitro biocompatibility and bioactivity of CPC/CSH/Sim/Col were performed with human dental pulp cells (hDPSCs). Moreover, in vivo evaluation was done using a dog animal model by micro-CT radiographic and histological analysis. The results shows that the developed CPC(CPC7CSH3), which contains 30 wt% CSH , exhibited optimal setting time and porous structure for clinical use. The cell viability and cytotoxicity exhibited that the CPC7CSH3 did not harm to the hDPSCs with or without contining Simvastatin or Collagenase. The animal study presents this CPC7/CSH3/Sim/Col biphasic biomaterial can induce dentin bridge formation. Based on the results, the developed CPC7/CSH3/Sim/Col biphasic biomaterial has great potential as a material for vital pulp therapy.