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)

摘要 数以百万计的人患有口腔干燥症，或由于缺乏唾液而导致的"口干"， 由于龋齿、口咽感染、吞咽困难（吞咽困难）增加而导致的生活质量 和消化（粘膜炎），味觉丧失和疼痛。再生医学可以提供创新策略 能够恢复患者的腺体功能，这些患者几乎没有选择。然而，目前缺乏 关于腺体再生机制和支架能力的基本科学知识， 促进这一过程，这仍然是治疗发展的一个重大限制。在之前的工作中，我们 开发了支持唾液附着、存活和顶基底极化的支架， 上皮细胞，这是分泌功能的要求。此外，微图案化的 具有半球形威尔斯孔的支架促进上皮细胞的结构和功能。因为分泌腺泡细胞 当原代小鼠下颌下腺上皮细胞在培养物中生长时， 在有或没有支架的情况下，我们研究了在体外培养的人骨髓基质细胞中对间充质细胞的需求。 保持它们的表型。初级唾液腺间充质细胞，但不是胚胎间充质细胞 系，在共培养物中维持腺泡分化。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 结合到支架中，可以支持腺泡分化。本申请提出了一种创新的， 多学科的策略来设计与多孔聚合物"海绵"集成的生物支架， 如底层，其将募集脉管系统并促进移植物的递送、存活和分化。 体内细胞我们假设纳米纤维支架具有间充质因子功能， 与海绵底层整合将能够移植祖细胞/前腺泡细胞， 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。 功能化的表皮细胞表面将递送上皮祖细胞并支持上皮细胞的保留。 原腺泡分化海绵与血管生成因子的功能化将招募和促进 血管网络的组装，以促进与宿主的整合和功能的有效再生。 体内组织。支架将在临床前小鼠唾液腺切除模型中进行测试，以检查 在体内支持组织再生的功效。将评估动物的唾液流量和唾液质量， 组织再生、新生长内细胞的分化状态以及再生组织的整合 与宿主血管系统的联系本文提出的使用小动物临床前模型的研究将为我们提供信息。 未来在大型动物模型中测试优化的支架，从而导致临床应用。 缩略语：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）、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- 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），FGF（成纤维细胞生长因子），FTIR（傅立叶变换红外光谱） 光谱学）、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-共-癸二酸酯））、PGSA（聚（甘油-共-癸二酸酯）-丙烯酸酯）、PLGA（聚乳酸-共-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）