研發搭載洛伐他汀之雙相型透明質酸膠體在牙髓組織再生的應用

Abstract

應用組織工程的理念，使用齒源性幹細胞並配合適當的支架材料，重建因感染而被移除的牙髓組織已成為活髓治療的新趨勢。然而誘導功能性牙本質與牙髓組織的再生並重建血流供應仍是目前牙髓組織再生研究的瓶頸。而雙相型透明質酸膠體以交聯型玻尿酸(crosslinked HA, cHA)為主要結構提供支架材料穩定性與強度，添加非交聯玻尿酸(non-crosslinked HA, ncHA)使其經酵素分解而具備釋放低分子量玻尿酸特性以調控細胞反應，並且可藉由調整交聯程度與膠體顆粒大小來控制材料具備可注射性質，非常適合用於牙髓組織再生醫療。因此本研究的目的是以可注射式雙相型透明質酸膠體為基礎，應用藥物釋放材料的理念，以PLGA奈米顆粒作為載體，搭載具有促進鈣化組織蛋白與血管增生因子表現效果的洛伐他汀(lovastatin)，研發新型的牙髓再生組織工程的支架材料。 使用1,4-丁二醇二縮水甘油醚（1,4-butanediol diglycidyl ether, BDDE）做為交聯劑製備濃度為2%而顆粒大小為420μm的交聯型透明質酸膠體顆粒（hyaluronic acid granules, HAG）。之後分別將重量比10%～40%之ncHA與HAG以物理性方法混合，製備雙相型透明質酸膠體顆粒（biphasic hyaluronic acid granules, biHAG）。接著再與洛伐他汀之聚乳酸聚甘醇酸奈米顆粒（PLGA-Lova）進行物理性混合，製備出搭載PLGA-Lova之雙相型透明酸膠體（Lova@biHAG）。並且進一步評估材料的物理性質、藥物釋放行為與生物相容性。 研究結果顯示，材料的BDDE殘留量皆小於FDA規定。隨著nHA添加比例增加，biHAG膨脹係數有明顯增加的趨勢，且其流體性質與可注射性都有明顯上升。而添加PLGA-Lova會略微提升材料的流動性，但對整體流變性質沒有顯著影響。其中流體性質最差的HAG100可以順利通過23G的針頭，而biHAG與Lova@biHAG組別則可使用25G針頭進行注射。顯微結構上，HAG與biHAG呈現多孔性蜂巢狀結構。相較於HAG100的結構，biHAG的組別可觀察到較細微的網狀結構，推測與nHA的添加有關。而Lova@biHAG與biHAG材料結構沒有明顯差異，可觀察到少許的PLGA-Lova粉末散佈在材料中。Lova@biHAG的藥物釋放行為和PLGA-Lova類似，但在總體藥物釋放量上，Lova@biHAG則僅有PLGA-Lova的六成。將材料與牙髓幹細胞（dental pulp stem cell, DPSC）共同培養一天的結果顯示，所有材料組別的細胞多呈現圓球或半球狀，僅有少部分細胞呈現伸出觸角的形態。HAG100和biHAG組的細胞表面完整，但Lova@biHAG組則可觀察到部分細胞表面出現微小破孔或是凹陷的缺損，推測可能與萃取液模型中測得過高的lovastatin釋放濃度有關。共同培養五天的觀察結果顯示，所有組別的細胞數量都有明顯增加，顯示biHAG適合DPSC細胞生長。使用材料萃取液與DPSC共同培養的結果發現，所有組別的材料在細胞存活率與死亡率的表現與控制組沒有顯著差異，都表現良好的生物相容性。但與大鼠細胞株（mineralizing rat pulpal cell line, MRPC-1）共同培養的細胞死亡率分析結果顯示，Lova@biHAG會造成約六成的細胞死亡率，明顯高於控制組與biHAG組，具有潛在毒性。 總結來說，本研究所研發之biHAG具有合適的流體性質與可注射性，同時表現出良好的生物相容性，適合DPSC生長分裂，具有應用於牙髓組織再生醫療的潛力。此外本研究建立混合PLGA-Lova和biHAG的方法並成功製備出Lova@biHAG材料，使biHAG不會影響PLGA-Lova所具有的藥物緩釋效果。但Lova@biHAG所釋放的藥物濃度過高，未來需要調整添加量降低釋放濃度以獲得促進牙本質再生與血管增生的效果。

Based on the principles of tissue engineering, the strategy to regenerate the damaged pulp tissue by using dental-derived stem cells with proper biomaterials as scaffold becomes the most potential treatment model in vital pulp therapy. However, regeneration of functional dentin and pulp tissue with vascularization is still a big challenge. The biphasic hyaluronic acid gel, containing cross-linked hyaluronic acid (cHA) as the major component for the strength and stability and non-crosslinked HA (ncHA) as additive for releasing small HA fragments to modulate cell function, could present the injectability via proper degree of cross-linkage and particle sizes, which are beneficial for pulp regeneration. In this study, we developed a novel scaffold for pulp regeneration based on biphasic hyaluronic acid granules (biHAG) with long-term and static statin releasing to stimulate the mineralization proteins and angiogenesis factor via drug delivery system of lovastatin using PLGA as carrier. The 2% cross-linked hyaluronic acid granules (HAG) with particle size of 420 μm were prepared via the crosslinking agent of 1,4-butanediol diglycidyl ether (BDDE). biHAG were prepared by physical mixing 10% to 40% (wt%) of ncHA with HAG, then polylactic-co-glycolic acid nanoparticles with lovastatin (PLGA-Lova) were added to obtain the biHAG with PLGA-Lova (Lova@biHAG). The physical properties, drug release behavior and biocompatibility of the materials were further investigated. The results showed the residual BDDE in HAG fitted to the FDA regulation. The swelling ratio, fluid properties and injectability of biHAG increased significantly when the ratio of ncHA increasing. The addition of PLGA-Lova slightly enhanced their fluid properties but no significant effects on the general rheological properties. In addition, biHAG and Lova@biHAG could be injected through 25G needle, but HAG100 only could be easy injected through 23G needle due to less fluid properties. HAG and biHAG presented the honeycomb microstructure. Compared with HAG100, a fine network structure related to nHA were observed in biHAG. Lova@biHAG presented the same microstructure with biHAG except few PLGA-Lova particles were observed. Lovastatin release behavior of Lova@biHAG were similar to that of PLGA-Lova, but only 60% of lovastatin releasing were found in Lova@biHAG compared to PLGA-Lova. After co-culture with dental pulp stem cells (DPSC) for 1 day, similar cell morphology was observed in all groups, in which most cells presented spherical or hemispherical shape and some with spread-out morphology. Unlike intact cell surface found in HAG100 and biHAG, the cells with defects of small holes and depression were observed in Lova@biHAG, which may relate to excessive concentration of lovastatin detected in extract model. After co-culture for 5 days, the cell numbers increased significantly in all groups, indicating biHAG were suitable for DPSC cell growth. Using extract model, no significant differences in cell viability and cell death of DPSC were found among all groups, proving the good biocompatibility of materials. However, the potential cytotoxicity of Lova@biHAG to mineralizing rat pulpal cell-1 (MRPC-1) was found by showing over 60% cell death compared to that of control and biHAG. In summary, biHAG presented the proper fluid properties and good injectablility and biocompatibility, indicating their potential using in pulp regeneration. In addition, we established a protocol to mix biHAG with PLGA-Lova and obtain Lova@biHAG successfully. No detrimental effect of biHAG on lovastatin release behavior of PLGA-Lova was found. But, excessive releasing of lovastatin of Lova@biHAG caused the potential toxicity. The proper mixing ratio of PLGA-Lova to biHAG should be further investigated in the future to stimulate dentin regeneration and angiogenesis.