Qi Fangfang, Yoshida Toshiko, Koike Takeshi, Aizawa Hitoshi, Shimane Tetsu, Li Yinghui, Yamada Shinichi, Okabe Motonori, Nikaido Toshio, Kurita Hiroshi
Department of Dentistry and Oral Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumato 390-8621, Japan.
Department of Regenerative Medicine, University of Toyama Graduate School of Medicine and Pharmaceutical Sciences, 2630 Sugitani, Toyama 930-0194, Japan.
Arch Oral Biol. 2016 May;65:26-34. doi: 10.1016/j.archoralbio.2016.01.011. Epub 2016 Jan 22.
Human amniotic membrane(HAM) as a graft material has been used in various fields. Hyper-dry amniotic membrane (HD-AM) is a novel dried amniotic membrane that is easy to handle and can be preserved at room temperature without time limitation. The purpose of this study was to investigate the useful properties of HD-AM in reconstruction of the oral mucosa.
Human oral keratinocytes were isolated and seeded on HD-AM in serum-free culture system. Oral mucosa equivalent (OME) was developed and transplanted onto full-thickness wound on athymic mice. The wound healing was analyzed and the OME both before and after transplantation was analyzed with hematoxylin-eosin staining and immunohistochemical staining for Cytokines 10 (CK10), Cytokines 16 (CK16), and Ivolucrin (IVL).
Oral keratinocytes spread and proliferated well on HD-AM. Two weeks after air-lifting, OME had formed with good differentiation and morphology. We confirmed immunohistochemically that the expression of CK10 was positive in all suprabasal layers, as was CK16 in the upper layers, while IVL was present in all cell layers. Three weeks after transplantation to athymic mice, the newly generated tissue had survived well with the smallest contraction. The epithelial cells of newly generated tissue expressed CK10 throughout in all suprabasal layers, IVL was mainly in the granular layer, and CK16 positive cells were observed in all spinous layer and granular layer but were not expressed in the mouse skin, all of which were similar to native gingival mucosa.
The OME with HD-AM as a matrix revealed a good morphology and stable wound healing. This study demonstrates that HD-AM is a useful and feasible biomaterial for oral mucosa reconstruction.
人羊膜（HAM）作为一种移植材料已被应用于各个领域。超干羊膜（HD-AM）是一种新型的干燥羊膜，易于操作，可在室温下无时间限制地保存。本研究的目的是探讨HD-AM在口腔黏膜重建中的有用特性。
分离人口腔角质形成细胞，并在无血清培养系统中接种于HD-AM上。构建口腔黏膜等效物（OME），并将其移植到无胸腺小鼠的全层伤口上。分析伤口愈合情况，并对移植前后的OME进行苏木精-伊红染色以及细胞角蛋白10（CK10）、细胞角蛋白16（CK16）和内披蛋白（IVL）的免疫组织化学染色分析。
口腔角质形成细胞在HD-AM上扩散并良好增殖。气提两周后，OME形成，具有良好的分化和形态。我们通过免疫组织化学证实，CK10在所有基底层以上各层均呈阳性表达，CK16在上层呈阳性表达，而IVL存在于所有细胞层。移植到无胸腺小鼠三周后，新生成的组织存活良好，收缩最小。新生成组织的上皮细胞在所有基底层以上各层均表达CK10，IVL主要在颗粒层，在所有棘层和颗粒层均观察到CK16阳性细胞，但在小鼠皮肤中不表达，所有这些都与天然牙龈黏膜相似。
以HD-AM为基质的OME显示出良好的形态和稳定的伤口愈合。本研究表明，HD-AM是一种用于口腔黏膜重建的有用且可行的生物材料。
