可注射温敏型水凝胶缓释Aspirin碳点和EPO促牙周组织再生的研究

Periodontitis, as one of the common oral diseases, is the main reason for the loss of teeth. The inflammation induced by periodontal pathogens results in the destruction of the periodontium. It has been proved that aspirin controls the inflammation by suppressing PI3K/Akt, ERK and NF-κB signalings. We previously found that the aspirin-based carbon dots possessed a better effect on anti-inflammation than that of pure aspirin; we also found that erythropoietin (EPO) promoted cell proliferation and bone formation. Therefore, it is promising to control inflammation by using aspirin-based carbon dots, and to promote periodontium regeneration by using EPO at the same time. To facilitate the repair of irregular defects, we will use an injectable and thermosensitive hydrogel as the carrier. We will prepare the hydrogels loaded with aspirin-based carbon dots and EPO; characterize the drug loading efficiency, encapsulating efficiency, controlled release and degradation. We will study the effects on anti-inflammation and osteoblast differentiation using cell and molecular biology techniques in vitro. The miniature pig model of periodontitis will be established for in vivo study. We will assess the effects on anti-inflammation and periodontium regeneration by micro-CT and histology. Our results will establish a method for treatment of periodontitis with drug loaded hydrogels, and lay the foundation for the clinical therapy of periodontitis.

牙周炎作为常见的口腔疾病之一，是引起牙脱落最主要的原因，牙周致病菌引起的炎症反应导致牙周组织的破坏。已有研究证明，阿司匹林（Aspirin）通过PI3K/Akt、ERK和NF-κB信号通路抑制炎症发生，我们前期研究发现，Aspirin碳点比普通阿司匹林具有更好的抗炎效果，且促红细胞生成素（EPO）能够促进细胞增殖和骨形成。因此，以Aspirin碳点抑制牙周炎症、EPO促进牙周组织再生有望治疗牙周炎。为便于不规则缺损腔隙的修复，我们以可注射温敏型水凝胶为载体，首先制备了温敏控释Aspirin碳点和EPO的水凝胶，表征其载药率、包封率、控制释放和降解能力；采用细胞生物学和分子生物学手段研究其对炎症的抑制作用及细胞的成骨向分化作用；建立小型猪牙周炎动物模型，通过micro-CT和组织学评价其抑制炎症、促进牙周组织再生效果。研究结果将建立一种治疗牙周炎的载药水凝胶体系，为牙周炎的临床治疗奠定基础。

牙周炎作为常见的口腔疾病之一，是引起牙脱落最主要的原因，牙周致病菌引起的炎症反应导致牙周组织的破坏。已有研究证明，阿司匹林（Aspirin）通过PI3K/Akt、ERK和NF-κB信号通路抑制炎症发生，且促红细胞生成素（EPO）能够促进细胞增殖和骨形成。因此，以Aspirin抑制牙周炎症、EPO促进牙周组织再生有望治疗牙周炎。为便于不规则缺损腔隙的修复，我们以可注射温敏型水凝胶为载体，制备了壳聚糖+β-甘油磷酸钠+明胶温敏型水凝胶，并将其与抗炎药物阿司匹林以及具有促再生作用的EPO复合，一方面控制牙周炎症，另一方面促进牙周组织再生。该水凝胶在37℃时凝胶化时间约为230s±15s，扫描电镜下观察，表面孔径约为40-80 μm，适合于细胞的长入和代谢废物的运出。药物释放检测，在前三天药物释放较快，阿司匹林和EPO释放量分别可达总量的86.6%和69.4%。随时间延长，药物持续释放至21天。为了观察体内成胶情况，将胶打到裸鼠和大鼠皮下，裸鼠5分钟内可以成胶。2周后大体观，背部皮下成胶周围没有明显的红肿坏死等炎症反应。HE切片显示实验组与对照组之间无明显差异，仅少量的炎细胞浸润。利用结扎丝在大鼠上颌第一磨牙建立了牙周炎模型，每3天给药1次，2周后Micro-CT结果显示：牙周炎组CEJ-AC间距离明显增大，双载组降低不明显，与空白对照组间无明显差异。组织学切片结果显示，空白对照组能够看到正常的牙周组织结构，完整的牙龈乳头和结合上皮。牙周炎组能够看到牙槽骨吸收明显，牙周炎组，水凝胶组和载EPO组可见大量的炎细胞浸润。载阿司匹林组和双载组炎症反应较轻。载EPO组和双载组可见折返线，说明骨改建比较活跃。IHC检测了COX-2和MMP-9的表达，也证明了载阿司匹林和双载组COX-2和MMP-9组阳细胞数明显减少，阿司匹林起到了较好的控制炎症的作用。说明所制备的水凝胶体系一方面能够抑制牙周炎症，另一方面能够促进牙周组织的再生。

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