Engineering Functioning Salivary Glands Using Micropatterned Scaffolds

摘要 数以百万计的人患有口干症，或“口干症”，由于缺乏唾液，产生的 因龋齿增加、口咽感染、吞咽困难(吞咽困难)而提高的生活质量 和消化(粘膜炎)、味觉丧失和疼痛。再生医学可以提供创新的策略 能够在几乎没有其他选择的患者中恢复腺体功能。然而，目前还缺乏 关于腺体再生机制和支架修复能力的基本科学知识 促进这一进程，这仍然是治疗学发展的一个实质性限制。在之前的工作中，我们 开发的纳米纤维支架支持唾液的附着、存活和根尖基底部极化 在体外培养上皮细胞，这是分泌功能所必需的。此外，微图案化 半球形孔支架促进了上皮细胞的结构和功能。因为分泌腺泡细胞 原代小鼠颌下腺上皮细胞在培养过程中也会丢失表型 在纳米纤维支架存在或不存在的情况下，我们研究了对间充质细胞的需求 保持它们的表型。原代唾液腺间充质细胞，但不是胚胎间充质细胞 株系，在共培养中保持腺泡分化。间充质因子能够替代 间充质维持原代上皮细胞的腺泡分化。这些间充质因子，当 整合到支架中，可以支持腺泡分化。该应用程序提出了一种创新的、 设计纳米纤维支架的多学科战略，这种支架与多孔性聚合物“海绵”整合在一起-- 类似于底层，将招募血管形成，促进移植的传递、存活和分化 体内的细胞。我们假设含有间充质因子的纳米纤维支架 与海绵底层相结合将使祖细胞/腺泡细胞移植成为可能 促进与宿主间充质和血管系统的整合，以恢复体内的唾液功能。 功能化的纳米纤维表面将传递上皮祖细胞，并支持保留 腺泡前分化。具有血管生成因子的海绵的功能化将招募和促进 组装血管网络，促进与宿主的整合和功能的有效再生 体内的组织。这些支架将在临床前小鼠唾液腺切除模型中进行测试，以检查 支持体内组织再生的功效。动物将接受唾液流量和唾液质量的评估， 组织再生，新生长中细胞的分化状态，以及再生组织的整合 与宿主血管系统的关系。这里提出的使用小动物临床前模型的研究将告诉我们 未来优化支架在大型动物模型中的测试，导致临床应用。 缩写：AQP5(水通道蛋白5)，DA(二丙烯酸酯)DAPI(4‘，6-二氨基苯基吲哚)，EC(内皮细胞 细胞)、E-钙粘素(E-钙粘素)、1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)、EMT(上皮-钙粘附素) 间充质转化)、表皮生长因子(EGF)、FACS(荧光激活细胞分选)、FFPE (福尔马林固定、石蜡包埋)、成纤维细胞生长因子(成纤维细胞生长因子)、傅立叶变换红外 光谱学)、H&E(苏木精-伊红)、ICC(免疫细胞化学)、IHC(免疫组织化学)、MA (甲基丙烯酸酯)、MACS(磁珠激活细胞分选)、Mx-ICC(多路免疫细胞化学)、 OCT(最佳切割温度化合物)，N-羟基琥珀酰亚胺(NHS)，聚乙二醇(PEGO)， PGS(聚二十二酸甘油共聚)、PGSA(聚甘油-癸二酸酯共聚)、PLGA(聚乳酸-乙二醇酸共聚) 酸性)、扫描电子显微镜(SEM)、颌下腺(SMG)、舌下腺(SLG)、紫外线 紫外线、血管内皮生长因子、血管内皮生长因子受体 2)、XPS(X射线光电子能谱)

ABSTRACT Millions of people suffer from xerostomia, or “drymouth” resulting from lack of saliva, producing a decreased quality of life due to increased dental caries, oropharyngeal infections, difficulties with swallowing (dysphagia) and digestion (mucositis), loss of taste, and pain. Regenerative medicine can offer innovative strategies capable of restoring gland function in patients that have few alternatives. However, there is a current lack of basic scientific knowledge regarding the mechanisms of gland regeneration and of the ability of scaffolds to promote this process, which remains a substantial limitation in development of therapeutics. In prior work, we developed nanofiber scaffolds that support the attachment, survival, and apicobasal polarization of salivary epithelial cells in vitro, which is a requirement for secretory function. Additionally, micropatterning of the scaffold with hemispherical wells promoted epithelial cell structure and function. Since the secretory acinar cell phenotype is lost when primary mouse submandibular salivary gland epithelial cells are grown in culture either in the presence or absence of nanofiber scaffolds, we investigated the requirement for mesenchymal cells in maintaining their phenotype. Primary salivary gland mesenchyme cells, but not an embryonic mesenchyme cell line, maintained acinar differentiation in co-cultures. Mesenchymal factors were able to substitute for the mesenchyme to maintain acinar differentiation of primary epithelial cells. These mesenchymal factors, when incorporated into a scaffold, may support acinar differentiation. This application proposes an innovative, multidisciplinary strategy to engineer nanofiber scaffolds that are integrated with a porous polymeric “sponge”- like underlayer that will recruit vasculature and facilitate delivery, survival and differentiation of transplanted cells in vivo. We hypothesize that a nanofiber scaffold functionalized with mesenchymal factors and integrated with a sponge underlayer will enable transplantation of progenitor/proacinar cells while facilitating integration with the host mesenchyme and vasculature to restore salivary function in vivo. The functionalized nanofiber surface will deliver the epithelial progenitor cells and support retention of proacinar differentiation. Functionalization of the sponge with angiogenic factors will recruit and facilitate assembly of vascular networks to promote integration with the host and effective regeneration of functional tissue in vivo. The scaffolds will be tested in a preclinical mouse salivary gland resection model to examine efficacy in supporting tissue regeneration in vivo. Animals will be assessed for salivary flow and saliva quality, tissue regrowth, differentiation state of cells within the new growth, and integration of the regenerated tissue with the host vascular system. The studies proposed here using a small animal preclinical model will inform future testing of an optimized scaffold in a large animal model, leading to clinical application. Abbreviations: Aqp5 (Aquaporin 5), DA (diacrylate) DAPI (4',6-diamidino-2-phenylindole), EC (endothelial cell), E-Cad (E-cadherin), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), EMT (epithelial- mesenchymal transition), epidermal growth factor (EGF), FACS (fluorescent activated cell sorting), FFPE (formalin-fixed, paraffin-embedded), FGF (fibroblast growth factor), FTIR (Fourier transform infrared spectroscopy), H&E (hematoxylin and eosin), ICC (immunocytochemistry), IHC (immunohistochemistry), MA (methacrylate), MACS (magnetic bead activated cell sorting), Mx-ICC (multiplexed immunocytochemistry), OCT (Optimal Cutting Temperature Compound), N-hydroxysuccinimide (NHS), PEG (Poly ethylene glycol), PGS (poly(glycerol-co-sebacate)), PGSA (poly(glycerol-co-sebacate)-acrylate), PLGA (Poly Lactic-co-Glycolic Acid), SEM (scanning electron microscopy), SMG (submandibular gland), SLG (sublingual gland), UV (ultraviolet), VEGF (vascular endothelial growth factor), VEGFR2 (vascular endothelial growth factor receptor 2), XPS (X-ray photoelectron spectroscopy)