Perlecan gradients in hydrogels for spatially-defined growth factor delivery

 描述（由申请人提供）： 口干症，或口干，是一种常见的和毁灭性的副作用，放射治疗通常用于治疗头颈部癌症患者。目前还没有治疗方法来恢复放射后的唾液功能，唯一可用的治疗方法是姑息性的。用于唾液腺再生的组织工程策略使细胞封装和存活成为可能，但不能完全复制在发育过程中观察到的复杂的3D图案。硫酸乙酰肝素蛋白聚糖（HSPG）和肝素结合生长因子（HBGF）的3D梯度在发育过程中建立了这些模式，并促进了适当的胚胎和成人组织和修复。水凝胶生物材料中的生长因子梯度仍然是组织工程研究人员的目标，但很少以复制天然组织的基质定向作用的方式产生。本文提出的研究的目标是在生物衍生的基于透明质酸（HA）的水凝胶内建立共价结合的HSPG的生物启发的3D梯度，采用这些来创建相关HBGF的互补梯度，并重现唾液腺的发育以促进有组织的组织再生。我们的假设是，HA水凝胶内的HSPG/HBGF梯度将使封装的原代人唾液细胞的形态和表型的受控、不对称发展成为可能，支持分支形态发生和导管形成。 在目标1中，将使用多通道装置和自动注射泵在HA水凝胶内产生来自HSPG串珠蛋白聚糖的共价结合串珠蛋白聚糖结构域1（PlnD 1）的梯度。水凝胶表征包括测定PlnD 1偶联效率和梯度分布。这些PlnD 1梯度的功能性HBGF结合能力将使用HBGF的标记形式如FGF-7和FGF-10测定，其促进唾液细胞分支形态发生。在目的2中，我们将评估几种PlnD 1和HBGF梯度分布对体外包封的人唾液腺腺泡衍生细胞（hSACs）的影响。将在整个培养过程中评估细胞表型、运动性和组织性，以响应HBGF梯度。在目标3中，将具有包封的hSAC的HBGF/PlnD 1梯度水凝胶植入唾液腮腺损伤的大鼠模型中并与对照进行比较。将在数周内通过功能性淀粉酶测定和组织学评分对唾液组织进行评价，以评估表型功能、植入物组织、与天然组织的整合和总体组织形态。 本研究将加深我们对HSPGs和形态发生素梯度对唾液组织再生的细胞反应的理解。此外，该研究将描述一个平台，该平台将广泛适用于再生医学应用中任何HBGF的梯度输送。

 DESCRIPTION (provided by applicant): Xerostomia, or dry mouth, is a common and devastating side effect of radiation therapy commonly used to treat head and neck cancer patients. No treatment currently exists to restore salivary function after radiation, and the only available therapies are palliative. Tissue engineering strategies for salivary gland regeneration enable cell encapsulation and survival, but do not fully replicate the intricate 3D patterning observed during development. 3D gradients of heparan sulfate proteoglycans (HSPGs) and heparin-binding growth factors (HBGFs) establish these patterns during development, and facilitate proper embryological and adult tissue organization and repair. Growth factor gradients within hydrogel biomaterials remain a target of tissue engineering researchers, but are rarely produced in a manner that replicates the matrix-directed action of native tissue. The goal of the research proposed herein is to establish biologically-inspired 3D gradients of covalently-bound HSPG within biologically-derived hyaluronic acid-based (HA) hydrogels, employ these to create complementary gradients of relevant HBGFs, and recapitulate the development of salivary glands to promote organized tissue regeneration. Our hypothesis is that HSPG/HBGF gradients within HA hydrogels will enable controlled, asymmetric development of morphology and phenotype of encapsulated primary human salivary cells, supporting branching morphogenesis and duct formation. In Aim 1, gradients of covalently-bound perlecan domain 1 (PlnD1) from the HSPG perlecan will be generated within HA hydrogels using multichannel devices and automated syringe pumps. Hydrogel characterization includes determination of PlnD1 coupling efficiency and gradient profiles. Functional HBGF binding capacity of these PlnD1 gradients will be determined using labeled versions of HBGFs such as FGF-7 and FGF-10, which promote salivary cell branching morphogenesis. In Aim 2, we will evaluate the effects of several PlnD1 and HBGF gradient profiles on encapsulated human salivary acinar-derived cells (hSACs) in vitro. Cell phenotype, motility, and organization will be assessed throughout culture in response to HBGF gradients. In Aim 3, HBGF/PlnD1 gradient hydrogels with encapsulated hSACs will be implanted in a rat model of salivary parotid gland damage and compared to controls. The salivary tissue will be evaluated via a functional amylase assay and histological scoring over several weeks to assess phenotypic function, implant organization, integration into native tissue, and overall tissue morphology. This research will enhance our understanding of cell response to gradients of HSPGs and morphogens for salivary tissue regeneration. Additionally, the research will describe a platform that will be broadly applicable for gradient delivery of any HBGF in regenerative medicine applications.