THE USE OF FIBRIN HYDROGELS TO PROMOTE SALIVARY GLAND REGENERATION

摘要 我们的工作到目前为止，在目前的赠款产生了以下结果：a）单个小鼠腮腺细胞， 发现形成显示TJ和激动剂诱导的分泌反应的三维（3D）细胞簇 当在生长因子减少的基质胶（GFR-MG）与纤维素水凝胶1（FH）的组合上生长时，B） GRF-MG和FH的比例进行了优化，最终目标是创建功能和临床安全的 支架1，c）我们的唾液细胞分离方法被改进以维持分泌颗粒，形成TJ， d）小鼠下颌下腺（mSMG）细胞，而不是腮腺或舌下腺， 被发现是产生显示有组织形态的唾液细胞簇的最佳选择，e）关键 在GFR-MG中鉴定了组分（即，EGF和IGF-1），增强唾液腺（SG） f）EGF和IGF-1被发现不能独立地诱导细胞分化。 产生有组织的细胞簇;然而，该目标是使用层粘连蛋白-1（L1）实现的，g）肽 （对应于促进完整SG形成的四个L1区）合成并缀合至FH，h） 特异性L1肽诱导大鼠腮腺细胞系Par-C10中腺泡球的形成（与 i）加入来自人毛囊间充质干细胞的条件培养基 （hHF-MSC CM）改善mSMG细胞中的唾液细胞簇组织和管腔形成， 簇维持腺泡、导管和肌上皮细胞，同时响应于分泌激动剂，k）管腔 1）在hHF-MSC CM中检测到大量的FGF 2， 其中生长完整唾液细胞簇的hHF-MSC CM，m）化学缀合至 将荧光FH应用于体内损伤的mSMG以形成新的唾液组织，与单独的FH相比 或者没有支架，小鼠在治疗后是健康的。总的来说，上述研究表明， 在这项赠款的头三年里，我们已经实现了我们最初的大部分目标， 所遇到困难的替代方案（例如，L1肽在增强FH和改善唾液细胞中的用途 如上所述的簇形成）。在即将到来的资助期内，我们将改进我们的设计， a）产生用L1肽和生长因子修饰的FH，B）生长分化 c）使用修饰的FH支架在体内形成新的功能性组织 (with目的是以后将这些发现应用于因舍格伦综合征而导致SG功能障碍的人， 头部和颈部γ辐射治疗）。

ABSTRACT Our work to date on the current grant has yielded the following results: a) single mouse parotid cells were found to form three-dimensional (3D) cell clusters displaying TJ and agonist-induced secretory responses when grown on Growth Factor-Reduced Matrigel (GFR-MG) in combination with Fibrin Hydrogel1 (FH), b) the ratio of GRF-MG and FH was optimized with the ultimate goal of creating a functional and clinically safe scaffold1, c) our salivary cell isolation method was improved to maintain secretory granules, form TJ and facilitate cell survival, d) mouse submandibular gland (mSMG) cells, rather than parotid or sublingual glands, were found to be the best choice for creating salivary cell clusters displaying organized morphology, e) critical components were identified within GFR-MG (i.e., EGF and IGF-1) that enhance salivary gland (SG) differentiation when polymerized to FH, f) EGF and IGF-1 were found to be incapable of independently producing organized cell clusters; however, this goal was achieved using Laminin-1 (L1), g) peptides (corresponding to four L1 regions that promote intact SG formation) were synthesized and conjugated to FH, h) specific L1 peptides induced formation of acinar spheres in the rat parotid cell line Par-C10 (as compared to cells grown on FH alone), i) addition of a conditioned medium from human hair follicle mesenchymal stem cells (hHF-MSC CM) improved salivary cell cluster organization and lumen formation in mSMG cells, j) salivary cell clusters maintained acinar, ductal and myoepithelial cells while responding to secretory agonists, k) lumen formation was impeded with the blocking of FGF10 in hHF-MSC CM, l) high amounts of FGF2 were detected in a hHF-MSC CM in which intact salivary cell clusters were grown, m) L1 peptides chemically conjugated to fluorescent FH were applied to wounded mSMG in vivo to form new salivary tissue, as compared to FH alone or no scaffold, and mice were healthy after the treatment. Taken as a whole, the above studies indicate we have accomplished the majority of our initial goals during the first three years of this grant and found viable alternatives for the difficulties encountered (e.g., use of L1 peptides to enhance FH and improve salivary cell clusters formation, as noted above). In the coming grant period, we will refine our design for a clinically safe environment as follows: a) produce a FH modified with L1 peptides and growth factors, b) grow differentiated salivary cell clusters in these matrices and c) use modified FH scaffold to form new and functional tissue in vivo (with aim of later applying these findings to humans with SG dysfunction due to Sjögren's syndrome as well as head and neck γ-irradiation treatments).