Directing Collective Epithelial Morphology in Space and Time Using a Light-Based Carving Tool

项目总结： 口干症，或“口干”，是一种极具挑战性的临床症状，由唾液细胞受损引起。 腺体。它可能是由各种组织损伤引起的，包括头部和头部放射治疗的急性损伤 宫颈癌，干燥病的进行性自身免疫反应，或其他因年龄增长而导致的不明原因。 目前的治疗方法只能暂时缓解症状，而且对相关的口腔健康的解决效果很差。 腐烂。这种情况的代价是相当大的，无论是在生活质量方面，还是在增加的经济负担方面 牙科护理。组织工程和再生医学领域为实现这一潜力提供了许多工具 在支持性水凝胶基质中重建健康的唾液来源细胞，但这些选择很少 提供足够的空间和时间分辨率，以恢复复杂的分支结构和精确的空间 主要唾液腺的表型图谱。然而，激光水凝胶降解方面的新发现 (LBHD)可以用来在完整的水凝胶片中“切割”路径，具有精确的亚细胞分辨率 XYZ，并提供了一种三维引导唾液上皮芽生长的方法。我们的假设是 目前的建议是，我们可以使用基于多光子的LBHD来拉长给定的多细胞簇 方向，并重新创造了本土腺体的关键元素。为此，我们将利用我们实验室的专业知识 从健康组织中分离原代人类唾液来源的干细胞/祖细胞(HS/PC)，以及 在可定制、生物兼容的透明质酸内封装为响应性3D多细胞球状簇 酸(HA)水凝胶。我们正在进行的工作表明，通过定制这些水凝胶的孔隙率和它们的 生物活性表位的浓度，我们可以影响簇的大小、形态以及与 周围的细胞外基质。我们将通过以下具体目标来验证我们的假设： 目的1.建立参数，通过羟基磷灰石水凝胶开凿“隧道”，促进HS/PC集群的生长。 目标2.使该系统适应于支持成纤维细胞共培养的替代基质，或结合耐光性 更容易制造的交联剂。目标3.评估正在成长的集群的表型，在其尾随和领先 边缘和分枝的末端，为分化表型的迹象。如果成功，这个系统可以作为一种 研究人类唾液细胞组织和分化机制的有用模型；该系统可能 也是制造组织工程化腺体替代物的早期原型。R03机制将 为必要的试点和可行性研究提供支持，以证明这些经过验证的技术 可以组合在一起生产出一种新颖的平台。

Project Summary: Xerostomia, or “dry mouth”, is a challenging clinical condition, caused by damage to the cells of the salivary gland. It may result from a variety of tissue insults, including acute damage from radiation therapy for head and neck cancers, progressive auto-immune response in Sjogren’s disease, or other unknown etiology from aging. Current treatments offer only temporary relief of symptoms, and poor resolution of associated oral health decay. The cost of this condition is considerable, both in quality of life and the financial burden of increased dental care. The fields of tissue engineering and regenerative medicine offer many tools for the potential reconstitution of healthy salivary-derived cells within supportive hydrogel matrices, but few of these options provide sufficient spatial and temporal resolution to restore the complex branched structure and precise spatial phenotype map of the major salivary glands. However, new discoveries in laser-based hydrogel degradation (LBHD) can be used to “carve” pathways through intact hydrogel slabs, with pinpoint, subcellular resolution in xyz, and offer a method to guide a growing salivary epithelial bud in 3 dimensions. Our hypothesis for the present proposal is that we can use multiphoton-based LBHD to elongate a multicellular cluster in a given direction, and recreate key elements of the native gland. To do this, we will employ our laboratory’s expertise in isolation of primary human salivary-derived stem/progenitor cells (hS/PCs) from healthy tissues, and encapsulation as responsive 3D multicellular spheroid clusters within customizable, biocompatible hyaluronic acid (HA) hydrogels. Our ongoing work has shown that, by tailoring the porosity of these hydrogels and their concentration of bioactive epitopes, we can impact cluster size, morphology, and interaction with the surrounding extracellular matrix. We will test our hypothesis through the following Specific Aims: Aim 1. Establish parameters to carve “tunnels” through HA hydrogels and promote HS/PC cluster ingrowth. Aim 2. Adapt the system to alternate matrices that enable fibroblast co-culture, or incorporate photolabile crosslinkers for easier fabrication. Aim 3. Assess phenotype of the growing cluster, at its trailing and leading edges and branched termini, for signs of differentiated phenotype. If successful, this system could serve as a useful model for studying mechanisms of human salivary cell organization and differentiation; the system might also be an early prototype for manufacturing tissue engineered gland replacements. The R03 mechanism will provide support for the necessary pilot and feasibility studies, to demonstrate that these proven technologies can be combined to produce a novel platform.