The Use of Fibrin Hydrogels to Build an Artificial Salivary Gland

描述（由申请人提供）：唾液在维持口腔健康方面起着重要作用。唾液分泌减少的患者（口腔干燥症）表现出咀嚼和吞咽食物困难、蛀牙、牙周病和微生物感染。尽管最近在治疗口干症方面有所改进，但很少有科学进展可以在临床上应用于恢复受损的唾液腺功能。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，这种恢复性治疗模型已被证明是不完整的，由于分化差和分泌机制的不良规范。我们相信，Fiorium水凝胶（FH）支架（在生长因子减少的基质胶[GFR-MG]中与生长因子和细胞外基质蛋白连接）将允许腺泡细胞分化并使得人工唾液腺的构建成为可能。我们已经尝试了两种方法来产生分化的腺泡结构（即，如高淀粉酶蛋白表达所证实）。GFR-MG允许腺泡细胞形成能够形成紧密连接（TJ）的有组织的三维（3D）结构;然而，基质本身是致瘤的。同样，FH是安全的，但在这种支架上生长的细胞不会形成3D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，当细胞在单独的GFR-MG或FH上生长时观察到的许多问题显然得到解决。具体地，形成3D腺泡结构（如单独的GFR-MG的情况）并且淀粉酶表达增加。然而，仍然存在的问题是，混合基质（GFR-MG/FH）仍然保留GFR-MG的致瘤性质，因此对于可植入腺泡结构的生长没有用。由于淀粉酶的产生是细胞分化的基本指标，我们打算研究在GFR-MG/FH上生长的腺泡3D构建体中增强淀粉酶表达和随后的极化顶端分泌的分子机制。我们的研究将确定GFR-MG和FH用于组织腺泡分化结构的最佳浓度，从而允许单个唾液腺泡细胞组织成分化的3D结构（目的1）。此外，我们将确定和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，我们将生长因子和细胞外基质蛋白掺入FH中（通过化学偶联和慢病毒基因递送），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 治疗策略，以恢复唾液腺功能障碍，有助于口腔干燥症的患者与受损的唾液腺功能。

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.