Perlecan gradients in hydrogels for spatially-defined growth factor delivery

 描述(申请人提供)：口干症，或口干，是一种常见的和破坏性的副作用，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辐射后的唾液功能，唯一可用的治疗方法是姑息性的。唾液腺再生的组织工程策略使细胞能够包裹和存活，但不能完全复制发育过程中观察到的复杂的3D图案。硫酸乙酰肝素蛋白多糖(HSPGs)和肝素结合生长因子(HBGFs)的三维梯度在发育过程中建立了这些模式，并促进了适当的胚胎学和成体组织和修复。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导向作用的方式产生。这项研究的目的是在生物衍生的透明质酸(HA)水凝胶中建立生物启发的共价结合HSPG的3D梯度，利用这些梯度来创建相关HBGFs的互补梯度，并概括唾液腺的发育以促进有组织的组织再生。我们的假设是，HA水凝胶中的HSPG/HBGF梯度将使包裹的原代人唾液细胞的形态和表型得到可控的、不对称的发展，支持分支形态发生和导管形成。在目标1中，来自HSPG Perlecan的共价结合Perlecan结构域1(PlnD1)的梯度将使用多通道设备和自动注射器泵在HA水凝胶中产生。水凝胶的表征包括PlnD1偶联效率和梯度分布的测定。这些PlnD1梯度的功能性HBGF结合能力将使用标记版本的HBGFs来确定，例如促进唾液细胞分支形态发生的hGFs-7和fGf-10。在目标2中，我们将评估几种PlnD1和HBGF梯度图谱对体外包裹的人唾液腺泡来源细胞(HSACs)的影响。在整个培养过程中，将根据HBGF梯度对细胞表型、运动性和组织进行评估。在目标3中，将HBGF/PlnD1梯度水凝胶包裹hSACs植入大鼠唾液腮腺损伤模型中，并与对照组进行比较。唾液组织将在几周内通过功能性淀粉酶检测和组织学评分进行评估，以评估表型功能、种植体组织、与自然组织的整合以及整体组织形态。这项研究将加深我们对唾液组织再生中HSPGs和形态原梯度的细胞反应的理解。此外，这项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的梯度递送。

 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.