The Use of Fibrin Hydrogels to Build an Artificial Salivary Gland

描述（申请人提供）：唾液在维持口腔健康中起着重要作用。唾液分泌减少的患者（症状表现为口干症）表现为咀嚼和吞咽食物困难、蛀牙、牙周病和微生物感染。尽管近年来在治疗口干症方面取得了进展，但在临床上用于修复受损唾液腺功能方面的科学进展很少。已经尝试推进恢复性治疗（通过使用各种细胞外基质开发人工唾液腺）。尽管如此，这种恢复性治疗模式已被证明是不完整的，因为缺乏分化和对分泌机制的不规范。我们相信纤维蛋白水凝胶（FH）支架（连接生长因子和细胞外基质蛋白在生长因子减少的基质中，[GFR- MG]）将允许腺泡细胞分化并使人工唾液腺的构建成为可能。我们尝试用GFR-MG和FH两种方法来产生分化的腺泡结构（即，作为淀粉酶蛋白高表达的证据）。GFR-MG使腺泡细胞形成有组织的三维（3D）结构，能够形成紧密连接（TJ）；然而，基质本身是致瘤性的。同样，FH是安全的，但是在这种支架上生长的细胞不能形成三维腺泡结构。此外，单独在GFR-MG或FH上生长的细胞不能达到完全分化，因此它们不能用于构建腺泡结构。有趣的是，当这些支架组合（GFR-MG/FH）时，细胞单独在GFR-MG或FH上生长时观察到的许多问题显然得到了解决。具体而言，形成三维腺泡结构（与单独使用GFR-MG的情况一样），淀粉酶表达增加。然而，混合基质（GFR-MG/FH）仍然保留GFR-MG的致瘤特性，因此不能用于可植入腺泡结构的生长，这仍然是一个问题。由于淀粉酶的产生是细胞分化的一个基本指标，我们打算研究在GFR- MG/FH上生长的腺泡3D构建体中，淀粉酶的表达和相应的尖分泌极化的分子机制。我们的研究将确定GFR-MG和FH组织腺泡分化结构的最佳浓度，使单个唾液腺泡细胞组织成分化的3D结构（目的1）。此外，我们将确定并分离负责腺泡细胞中淀粉酶表达的GFR-MG生长因子（目的2）。最后，我们将把生长因子和细胞外基质蛋白固定在FH中（通过化学偶联和慢病毒基因传递），以评估它们对唾液腺泡分化和分泌功能的影响（目的3）。这些研究应该会带来更好的结果

DESCRIPTION (provided by applicant): Saliva plays a major role in maintaining oral health. Patients with decreased saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease and microbial infections. Despite recent improvements in treating xerostomia, few scientific advancements have occurred which can be clinically applied toward restoration of compromised salivary gland function. Attempts have been made to advance restorative treatments (via development of an artificial salivary gland using a variety of extracellular matrices). Nonetheless, such restorative treatment models have proven incomplete due to poor differentiation and poor specification of mechanisms underlying secretion. We believe a Fibrin hydrogel (FH) scaffold (linked to growth factors and extracellular matrix proteins in growth factor-reduced-Matrigel, [GFR- MG]) will allow acinar cells to differentiate and make possible construction of an artificial salivary gland. We have tried two approaches to produce differentiated acinar structures (i.e., as evidence by high amylase protein expression), using GFR-MG and FH. GFR-MG allows acinar cells to from organized three- dimensional (3D) structures capable of developing tight junctions (TJ); however, the matrix itself is tumorogenic. Likewise, FH are safe, but cells grown on this scaffold do not form 3D acinar structures. Moreover, cells grown on either GFR-MG or FH alone do not reach full differentiation, so they cannot be used to build an acinar structure. Interestingly, whe these scaffolds are combined (GFR-MG/FH), many of the issues observed when cells are grown on GFR-MG or FH alone apparently are resolved. Specifically, a 3D acinar structure is formed (as was the case with GFR-MG alone) and amylase expression is increased. It remains a problem, however, that the hybrid matrix (GFR-MG/FH) still retains tumorogenic properties of GFR- MG and thus is not useful for growth of an implantable acinar structure. Because amylase production is a fundamental indicator of cell differentiation, we intend to investigate the molecular mechanisms that enhance amylase expression and consequent polarized apical secretion in acinar 3D constructs grown on GFR- MG/FH. Our studies will determine the optimal concentration of GFR-MG and FH for organization of acinar differentiated structures allowing single salivary acinar cells to organize into differentiated 3D structures (Aim 1). Additionally, w will determine and isolate the GFR-MG growth factors responsible for amylase expression in acinar cells (Aim 2). Finally, we will immobilize growth factors and extracellular matrix proteins into FH (by chemical conjugation and lentiviral gene delivery) to evaluate their effects on salivary acinar differentiation and secretory function (Aim 3). These studies should lead to better therapeutic strategies to restore salivary gland dysfunction that contributes to xerostomia in patients with compromised salivary function.