研發活髓治療運用之生物活性聚對二甲苯孔洞性支架

Development of bioactive parylene porous scaffold for vital pulp therapy

葉筱雯(Hsiao-Wen Yeh)

指導教授 ： 姜昱至

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103701

摘要

背景：
活髓治療泛指施行在具有可回復性的牙髓傷害之牙齒上，以維持牙髓活性與功能的術式 [1]。其目的為給予保護性的覆蓋材料以阻絕感染、避免進一步的傷害、維持牙髓活性，進而有助於牙髓組織之癒合及修復 [2]。活髓治療的成功經常伴隨著硬組織的生成，因此鈣化組織屏障或是類牙本質的形成常被認定為牙髓再生成功的一個現象 [3]，雖然該現象非治療之最終目的，但多數的實驗常用鈣化組織或類牙本質組織生成來作為牙髓組織修復或再生的判斷依據。目前臨床上可用來進行活髓治療材料的種類有幾種，如氫氧化鈣、三氧礦化物、Biodentine、其他生物陶瓷等，但綜觀各材料之特性，至今仍缺乏理想之活髓治療材料。故本研究為研發可應用於活髓治療之可攜帶生物因子之生物活性聚對二甲苯孔洞性支架。
材料及方法：
攜帶生長因子的聚對二甲苯支架 (PPxS-GFs) 通過化學氣相沉積聚合製造而成。對於支架非活體的測試包括通過電腦斷層掃描 (micro-CT) 評估是否有攜帶Wnt3a 和 FGF-2 的聚對二甲苯支架對於三維 (3-D) 顯微結構是否會有影響。另一方面將攜帶或無攜帶 Wnt3a 和 FGF-2 的支架與牙髓幹細胞 (DPSC) 一起培養，並通過Live and dead stain以及 MTT 測定來測試生物相容性。進一步透過標記骨鈣素（OCN）和牙本質唾液酸磷蛋白（DSPP）的表現，在免疫螢光染色下來確認誘導分化成牙本質母細胞的能力。活體實驗的部分，雄性Wistar大鼠的上頜第一大臼齒進行窩洞備製至牙髓暴露，暴露的牙髓分別使用玻璃離子體（對照組）、PPxS、PPxS 加生長因子（PPxs-GFs）、PPxS 加 DPSC（PPxS/DPSC）、PPxS 加生長因子和 DPSC（PPxS-GFs/DPSC）覆蓋，冠部窩洞使用玻璃離子體填補。大鼠於1、2、4、8週之時間點犧牲，後續進行電腦斷層、組織學和免疫組織化學分析。
結果與討論：
PPxS 和 PPxS-GFs 擁有相近的孔徑和孔隙率。 PPxS 和 PPxS-GFs 顯示出良好的生物相容性，而添加了Wnt3a及FGF-2的PPxS對於細胞增生有著較好的效果。進一步的測試PPxS-GFs對於牙本質母細胞分化的影響，顯示出PPxS-GFs較控制組有較佳促使牙髓幹細胞分化為牙本質母細胞的能力。動物研究的Micro-CT分析顯示PPxS/DPSC、PPxS-GFs 和 PPxS-GFs/DPSC於牙髓組織下方可見礦物化組織。 組織學檢查PPxS/DPSC 4週和PPxS-GFs/DPSC 4週和8 週的組別在牙髓組織中覆髓材料下誘導了礦物化組織生成。
結論：
Wnt3a 和 FGF-2 的添加不影響聚對二甲苯支架的結構及孔隙率，且聚對二甲苯支架與牙髓幹細胞之間具有良好的生物相容性。含有 Wnt3a 和 FGF-2的聚對二甲苯支架的在非活體實驗中，可誘導牙髓幹細胞分化為牙本質母細胞。 PPsX/DPSC、PPsX/GFs、PPsX-GFs/DPSC 在動物實驗中，可誘導生成覆髓材料下方的礦物化組織。

關鍵字

活髓治療 ； 覆髓 ； 聚對二甲苯 ； 動物實驗 ； 生長因子

並列摘要

Background: Maintaining pulp vitality during restorative and endodontic procedures is a recently emerged concept that has been termed vital pulp therapy (VPT). This involves covering the pulpal surface with a dental material to facilitate the formation of reparative dentin [4]. Both the research on VPT and the related clinical techniques have advanced. However, the material currently used in vital pulp therapy has some limitations. Thus, the purpose of this research was to develop novel biomaterials, specifically a designable, growth factor and stem cells carriable 3-D porous parylene scaffold (PPxS), for use in vital pulp therapy.
Materials and methods: A parylene scaffold carrying growth factors (i.e., Wnt3a and FGF-2), which was termed PPxS-GFs, was fabricated via chemical vapor deposition polymerization. The three-dimensional (3-D) microstructural and architectural features of PPxS, both with and without Wnt3a and FGF-2, were assessed using micro-computed tomography (micro-CT). The scaffolds with and without Wnt3a and FGF-2 were used to culture dental pulp stem cells (DPSCs), and biocompatibility was assessed via live and dead staining and the MTT assay. Induction of odontoblast differentiation was assessed by osteocalcin (OCN) labeling and evaluation of dentin sialophosphoprotein (DSPP) expression and was confirmed via immunofluorescence staining. The maxillary molars of male Wistar rats were pulp exposed and capped for 1, 2, 4, and 8 weeks with GI (control), PPxS, PPxS with growth factors (PPxs-GFs), PPxS with DPSCs (PPxS/DPSC), or PPxS with growth factors and DPSCs (PPxS-GFs/DPSC), and then sealed with GI. Then, the teeth were evaluated using non-destructive micro-CT and histologic and immunohistochemical analyses.
Results: The results revealed that the pore size and porosity of PPxS and PPxS-GFs were similar and both showed good cell viability and biocompatibility. However, the PPxS-treated pulp showed significantly higher cell proliferation. Micro-CT analysis of the study animals showed mineralized tissue formed beneath the cavity in the pulp tissue in the PPxS/DPSC, PPxS-GFs, and PPxS-GFs/DPSC groups. Histologic examinations showed tertiary dentin formation in the pulp tissue beneath the capping agent in the PPxS/DPSC group at 4 weeks and the PPxS-GFs/DPSC group at 4 and 8 weeks.
Conclusion: The addition of Wnt3a and FGF-2 did not affect the structure or porosity of the parylene scaffold. The parylene scaffold had good biocompatibility with DPSCs. The addition of Wnt3a and FGF-2 in the parylene scaffold induced the DPSCs to differentiate into odontoblasts in vitro. The PPxS/DPSC, PPxS/GFs, and PPxS-GFs/DPSC scaffolds induced mineralized tissue formation beneath the capping materials in vivo.

並列關鍵字

vital pulp therapy ； capping ； parylene ； animal study ； growth factor

參考文獻

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