研發奈米硫酸鈣/氫氧基磷灰石雙相生醫材料攜帶TGF-β1/VEGF於活髓治療之應用－材料性質、生物相容性及動物實驗

Abstract

活髓治療的目的在於治療任何可回復性的牙髓傷害，給予具生物活性與保護性之覆髓材料，並保持牙髓活性與誘導牙本質－牙髓修復再生。然而，活髓材料發展至今，在臨床上仍然沒有理想之活髓材料。目前在臨床上較常使用的活髓材料存在著操作性質差、硬化時間過長、機械性質不足或誘導牙髓–牙本質再生能力不足等缺點。為了改良並取代目前活髓材料的不足，本研究以奈米硫酸鈣和氫氧基磷灰石為基質，研發出可攜帶複方生長因子 (TGF-β1/VEGF) 之雙相生醫活髓材料。 本研究主要分成四大部分進行：第一部分為材料製成、物理性質評估與最佳化之研究。利用低溫真空法將二水硫酸鈣製成奈米級硫酸鈣，並利用化學共沉澱法合成氫氧基磷灰石，與醫療級半水硫酸鈣三種材料混合調整出最佳比例後，進行材料物理性質測試，包括以X光繞射分析材料結晶相、掃瞄式電子顯微鏡觀察材料表面結構、硬化時間、材料降解度、抗壓強度測試、以及生長因子釋放曲線分析等。第二部分為分析醫材體外細胞毒性與生物相容性；第三部分主要是分析材料誘導牙髓細胞之礦化能力，以評估其當活髓材料在誘導牙本質－牙髓組織再生中所扮演的角色。第四部分則以大鼠作為模型進行動物實驗、微電腦斷層(Micro-CT)非破壞性檢視，以及組織形態學分析。 結果顯示，雙相複合材料 nCS/HAp/CS 的初始硬化時間為 15 分鐘，符合臨床操作性質。浸泡在磷酸緩衝溶液中的複合材料 1 天的抗壓強度可維持在 7.0 MPa 左右，並在 1 週後有下降的趨勢。生長因子 (TGF-β1/VEGF) 在 14 天內可測得 60∼70 %的釋放量。生物相容性測試結果顯示雙相複合材料並不會對人類牙髓細胞的增生或存活造成影響。在雙相材料中加入 6 ng/mL TGF-β1 和 0.5 ng/mL VEGF 亦可促進鹼性磷酸酶和細胞基質礦化小體的表現。而動物實驗方面，以攜帶 TGF- β1 和 VEGF 的雙相生醫材料活髓治療 4 週後之大鼠臼齒，藉由 Micro-CT 和組織切片觀察到明顯的修復性牙本質橋形成。 綜合以上結論，顯示以本實驗所研發之奈米硫酸鈣與氫氧基磷灰石雙相生醫材料攜帶 TGF- β1 和 VEGF，在操作性質、生物相容性、促進細胞礦化能力，以及誘導牙本質－牙髓修復再生之能力均不亞於市售產品之表現，故在臨床應用上對於活髓治療之發展應具有相當的潛力。

To treat the reversible pulpal injury, vital pulp therapy aims to preserve pulp vitality as well as to induce dentin-pulp repair/regeneration by direct applying a bioactive material. However, after years of research and development of the capping agents, there is still a shortage of the ideal capping material for vital pulp therapy. Current pulp capping materials present several limitations, such as poor handling properties, prolonged setting time, insufficient mechanical properties, or difficulty to induce dentin-pulp complex regeneration, which in turn influences the prognosis of vital pulp therapy. Hence, the innovation of vital pulp therapy is important to overcome the limitations of current materials. The purpose of this study is to develop a biphasic biomaterial with inorganic materials, nano-calcium sulfate (nCS) and hydroxyapatite (HAp) as a bioactive pulp capping agent which is able to carry dual growth factors (TGF-β1/VEGF) for the induction of dentin-pulp complex regeneration. This study carried out in four parts-Part I: By using cryo–vacuum method, calcium sulfate dihydrate (CSD) was modified to nano-calcium sulfate hemihydrate. Hydroxyapatite was synthesized via chemical co-precipitation. We characterized crystalline phase via X-ray diffraction, and observed the microstructure by scanning electron microscope. The physical properties such as the setting time, compressive strength, in vitro degradation rate and the releasing profile of growth factors were determined while the ratio of nCS/HAp/CS was optimized. Part II: The cell-material interaction study, WST-1 and LDH assay was performed to evaluate the biocompatibility and cell cytotoxicity of nCS/HAp/CS/TGF-β1/VEGF cement. Part III: Evaluation of the cell mineralization ability of human dental pulp cell in response to the biomaterials so as to assess their roles in the pulp capping material in term of the induction of dentin-pulp complex regeneration. Part IV: An animal model of rat was established. In vivo evaluation was performed by non-destructive micro-CT determination and histological analysis. The results revealed that the initial setting time of the nCS/HAp/CS biphasic cement was 15 minutes, which exhibits substantial improvement compared with the commercial product. The compressive strength of the biphasic cement after immersion in phosphate buffered saline solution in 1 day was 7.0 MPa, and decreased after 1 week. Sustained release of TGF-β1/VEGF was achieved up to 60~70% through the biomaterial within 2 weeks. The excellent biocompatibility of the biphasic cement was also confirmed by WST-1 and LDH test, which indicates no significant difference between control group (DMEM) and experimental group (nCS/HAp/CS/TGF-β1/VEGF). Pulp cells treated with nCS/HAp/CS carrying 6 ng/mL TGF-β1 and 0.5 ng/mL VEGF showed increased level of ALP activity and formation of calcified nodules in vitro. In the animal study, the non-destructive determination with micro-CT evaluation showed hard tissue formation during 4-week measurements. Histological analysis demonstrated a prominent formation of reparative dentin bridge with nCS/HAp/CS/TGF-β1/VEGF cement after 4 weeks. Based on the present findings, in view of the advantages of the nCS/HAp/CS/TGF-β1/VEGF cement as the bioactive dressing material for vital pulp therapy, such novel compound is as competitive as the commercial products in minimally invasive treatment of dental pulp therapy and has promising prospects. We concluded that the developed nCS/HAp/CS cement could act as a potent carrier for sustained release of growth factors, and the nCS/HAp/CS/TGF-β1/VEGF cement is of great potential to promote dentin-pulp complex regeneration.