THE USE OF FIBRIN HYDROGELS TO PROMOTE SALIVARY GLAND REGENERATION

摘要 到目前为止，我们关于当前赠款的工作已经产生了以下结果：a)单个小鼠腮腺细胞 发现形成三维(3D)细胞团，显示TJ和激动剂诱导的分泌反应 当生长在生长因子还原基质(GFR-MG)和纤维蛋白水凝胶(FH)上时，b) 优化GRF-MG和FH的比例，最终目标是创建一个功能和临床安全的 支架1，c)改进了唾液细胞分离方法，以保持分泌颗粒，形成TJ和 促进细胞存活，d)小鼠下颌下腺(MSMG)细胞，而不是腮腺或舌下腺， 被发现是创建显示有组织形态的唾液细胞团的最佳选择，e)关键 在GFR-MG中发现了增强唾液腺的成分(即EGF和IGF-1)。 当聚合为FH时，发现EGF和IGF-1不能独立分化 产生有组织的细胞团；然而，这一目标是使用层粘连蛋白-1(L1)，g)肽实现的 (对应于促进完整SG形成的四个L1区域)被合成并与Fh，h结合) 特异性L1肽诱导大鼠腮腺细胞系PAR-C10形成腺泡球体(与 仅在FH上生长的细胞)，i)添加来自人毛囊间充质干细胞的条件培养液 (HHF-MSC CM)改善mSMG细胞的唾液细胞团组织和管腔形成，j)唾液细胞 集群维持腺泡、导管和肌上皮细胞，同时对分泌激动剂k)腔做出反应 在HHF-MSC CM中FGF10的形成被阻断)，L)在HHF-MSC CM中检测到大量的FGF2 一种HHF-MSC CM，在其中培养完整的唾液细胞簇，m)L1肽化学连接到 与单独应用FH相比，将荧光FH应用于体内损伤的mSMG形成新的唾液组织 或无支架，治疗后小鼠健康。总体而言，上述研究表明，我们 在这笔赠款的前三年完成了我们最初的大部分目标，并发现可行 解决遇到的困难的替代方案(例如，使用L1肽来增强FH和改善唾液细胞 形成集群，如上所述)。在即将到来的拨款期间，我们将改进我们的设计，以实现临床安全 环境如下：a)产生用L1肽和生长因子修饰的FH，b)生长分化 C)使用改良的FH支架在体内形成新的有功能的组织 (目标是稍后将这些发现应用于因Sjögren综合征和 头颈部γ照射治疗)。

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).