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方面起着重要作用。唾液分泌减少(症状为口干症)的患者表现出咀嚼和吞咽食物困难、龋齿、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，但几乎没有科学进展可以应用于临床恢复受损的唾液腺功能。人们试图推进恢复性治疗(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，因为分化较差，而且对潜在的分泌机制没有很好的说明。我们相信，纤维蛋白水凝胶(FH)支架(连接到生长因子和生长因子还原Matrigel中的细胞外基质蛋白，[GFR-MG])将允许腺泡细胞分化，并使构建人工唾液腺成为可能。我们已经尝试了两种方法来产生分化的腺泡结构(即，通过高淀粉酶蛋白表达来证明)，使用GFR-MG和FH。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，基质本身是致癌的。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，单独生长在GFR-MG或FH上的细胞不能达到完全分化，因此它们不能用来构建腺泡结构。有趣的是，当这些支架结合在一起(GFR-MG/FH)时，当细胞单独生长在GFR-MG或FH上时，观察到的许多问题显然得到了解决。具体地说，形成了三维腺泡结构(就像GFR-MG单独的情况一样)，并且淀粉酶的表达增加。然而，这仍然是一个问题，杂化基质(GFR-MG/FH)仍然保留着GFR-MG的致瘤特性，因此不利于植入性腺泡结构的生长。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结构中促进淀粉酶表达并随后极化顶端分泌的分子机制。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(目标1)。此外，w将确定并分离导致腺泡细胞中淀粉酶表达的GFR-MG生长因子(目标2)。最后，我们将通过化学偶联和慢病毒基因传递将生长因子和细胞外基质蛋白固定到FH中，以评估它们对唾液腺泡分化和分泌功能的影响(目标3)。这些研究应该会带来更好的结果 恢复唾液腺功能障碍的治疗策略，这种功能障碍会导致唾液功能受损患者的口干症。

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.