Engineering Functioning Salivary Glands Using Micropatterned Scaffolds

使用微图案支架工程功能唾液腺

• 批准号: 9507143
• 负责人: James Castracane
• 金额: $ 65.27万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9507143
• 关键词: Abbreviations; Acinar Cell; Acrylates; Address; Adverse effects; Angiogenic Factor; Animal Model; Animals; Apical; Basement membrane; Biological Assay; Blood Vessels; Caliber; Carbodiimides; Cell Differentiation process; Cell Line; Cell Separation; Cell Transplants; Cell physiology; Cells; Cellular Structures; Chemicals; Clinical; Coculture Techniques; Collaborations; Deglutition; Deglutition Disorders; Dental caries; Development; Devices; Digestion; E-Cadherin; Elastin; Electrospinning; Embryo; Endothelial Cells; Engineering; Epidermal Growth Factor; Epithelial; Epithelial Cells; Epithelium; Excision; Fibroblast Growth Factor; Formalin; Future; Gland; Glass; Glycerol; Glycolates; Goals; Hematoxylin and Eosin Staining Method; Hybrids; Immunohistochemistry; In Vitro; Infection; Joints; KDR gene; Knowledge; Laminin; Link; Manuscripts; Mesenchymal; Mesenchyme; Methacrylates; Methods; Modeling; Mucositis; Mus; Nanotopography; Natural regeneration; Oral; Organ; Oropharyngeal; Pain; Palliative Care; Paraffin Embedding; Patients; Peer Review; Phenotype; Polymers; Porifera; Pre-Clinical Model; Preparation; Process; Production; Property; Publications; Publishing; Quality of life; Radial; Recruitment Activity; Regenerative Medicine; Research; Roentgen Rays; Saliva; Salivary; Salivary Glands; Scanning Electron Microscopy; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Stem cells; Structure; Sublingual Gland; Submandibular gland; Surface; Surface Properties; Symptoms; System; Taste Perception; Temperature; Testing; Therapeutic; Tight Junctions; Tissues; Transplantation; United States; Vascular Endothelial Growth Factors; Vascular System; Vascularization; Work; Xerostomia; aquaporin 5; base; clinical application; clinical translation; ethylene glycol; experience; immunocytochemistry; improved; in vivo; in vivo regeneration; innovation; magnetic beads; mechanical properties; multidisciplinary; nanofiber; new growth; occludin; pre-clinical; preclinical study; progenitor; reduce symptoms; regenerative; saliva secretion; salivary acinar cell; scaffold; software development; therapeutic development; tissue regeneration; ultraviolet; water channel

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)

抽象的 数百万人患有口干症，或因缺乏唾液而导致的“口干症”，导致唾液分泌减少 由于龋齿增加、口咽感染、吞咽困难（吞咽困难）而导致生活质量下降 消化（粘膜炎）、味觉丧失和疼痛等再生医学可以提供创新策略。 能够恢复几乎没有其他选择的患者的腺体功能，但目前缺乏。 有关腺体再生机制和支架能力的基本科学知识 在之前的工作中，我们促进了这一过程，这仍然是治疗学发展的一个重大限制。 开发了支持唾液附着、存活和顶端基底极化的纳米纤维支架 体外的上皮细胞，这是分泌功能的要求。 具有半球形孔的支架促进了分泌性腺泡细胞的上皮细胞结构和功能。 当原代小鼠颌下唾液腺上皮细胞在培养物中生长时，表型会丢失 在存在或不存在纳米纤维支架的情况下，我们研究了对间充质细胞的需求 维持原代唾液腺间充质细胞的表型，但不是胚胎间充质细胞。 线，在共培养中维持腺泡分化，间充质因子能够替代。 间充质维持初级上皮细胞的腺泡分化。 结合到支架中，可以支持腺泡分化。该应用提出了一种创新的、 多学科策略来设计与多孔聚合物“海绵”集成的纳米纤维支架 - 就像底层一样，它将招募脉管系统并促进移植物的输送、存活和分化 我们捕获了具有间充质因子功能的纳米纤维支架 与海绵底层集成将能够移植祖细胞/原腺细胞，同时 促进与宿主间充质和脉管系统的整合，以恢复体内唾液功能。 功能化纳米纤维表面将输送上皮祖细胞并支持保留 具有血管生成因子的海绵的功能化将募集并促进。 血管网络的组装，促进与宿主的整合和功能性血管的有效再生 支架将在临床前小鼠唾液腺切除模型中进行测试以进行检查。 将评估动物体内支持组织再生的功效。 组织再生、新生长的细胞的分化状态以及再生组织的整合 这里提出的使用小动物临床前模型的研究将提供信息。 未来在大型动物模型中测试优化的支架，从而实现临床应用。 缩写：Aqp5（水通道蛋白 5）、DA（二丙烯酸酯）、DAPI（4',6-二脒基-2-苯基吲哚）、EC（内皮细胞） 细胞）、E-Cad（E-钙粘蛋白）、1-乙基-3-（3-二甲氨基丙基）碳二亚胺盐酸盐（EDC）、EMT（上皮细胞） 间质转化）、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）、FGF（成纤维细胞生长因子）、FTIR（傅里叶变换红外） 光谱法）、H&E（苏木精和伊红）、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（多重免疫细胞化学）、 OCT（最佳切削温度化合物）、N-羟基琥珀酰亚胺（NHS）、PEG（聚乙二醇）、 PGS（聚（甘油-癸二酸酯））、PGSA（聚（甘油-癸二酸酯）-丙烯酸酯）、PLGA（聚乳酸-乙醇酸） 酸）、SEM（扫描电子显微镜）、SMG（颌下腺）、SLG（舌下腺）、UV （紫外线）、VEGF（血管内皮生长因子）、VEGFR2（血管内皮生长因子受体） 2）、XPS（X射线光电子能谱）