Multiscale modeling of an inductive hair follicle microenvironment in engineered skin substitute

如果符合以下条件，工程皮肤组织必须复制其天然同类的生物和机械功能 他们将为社会提供健康福利。然而，工程皮肤替代物(ESS)仅适用于基本皮肤 功能与人体皮肤的结构和生物物理特征不符，如缺失 毛囊和汗腺，限制了其在体内的使用。它们的缺席是由于缺乏功能正常的细胞类型 这种指导人类角质形成细胞在ESS中形成毛囊的研究还缺乏深入的了解 推动毛囊形成的基本上皮-间充质相互作用。以毛囊为例 工程学，角质形成细胞与其直接真皮之间的上皮-间充质相互作用 需要精确地调节环境以控制毛囊的世系承诺。主要的细胞类型是 构成一种独特的真皮“利基”是一种特殊的成纤维细胞群体，它们位于 毛囊称为毛乳头(DP)，不同于正常的人类真皮成纤维细胞。然而， 在体外分离和扩增人DP成纤维细胞，同时保持其诱导能力是非常困难的 用于组织工程目的。长期目标是开发新的生物工程方法来生产 一种功能齐全的人体皮肤，具有正常的显微解剖。中心假设是毛囊 诱导是皮肤结构的一种紧急属性，它需要多种信号和细胞的相互作用 在诱导性微环境中的类型。目标是系统地探索如何创建一个归纳的 ESS中诱导毛囊形成的微环境。为了实现这一目标，我们制定了一个三管齐下的方案 解决毛囊生物工程在ESS中的策略：1)通过制造复合材料来模拟原始的壁龛 微图案化ESS中角质形成细胞-DP细胞球体；2)通过添加Bmp6和Bmp6增强细胞间相互作用 3)通过重新激活主转录因子来驱动DP表型。在第一个目标中，我们将确定是否有多个 细胞型球体与预制的毛管相结合，可以模拟ESS中的自然利基环境。我们已经开发出 两个3D复合球体模型，并将确定它们是否允许DP成纤维细胞在体外扩张 同时在激光微图案化皮肤替代模型中保持DP诱导性以诱导毛囊。在 第二个目的，我们将确定Bmp6在ESS中刺激毛囊形成的诱导作用。我们 将剖析Bmp6在毛囊形成和生长中的作用。在第三个目标中，我们将确定是否 人的DP成纤维细胞可以被重新编程以重建毛发诱导能力。我们将评估体外遗传基因 CRISPRA介导的主转录因子表达对人DP成纤维细胞的重编程作用 促进复合角质形成细胞-DP细胞球体在ESS中诱导毛囊的能力。这项工作将 提供关于空间生态位排列、感应信号和遗传程序的新的机械知识 是促进ESS中毛囊新生的关键因素。结果很重要，因为它会加速头发的生长 卵泡生物工程和体内、体外组织工程及再生医学的先进复合体。

Engineered skin tissues must reproduce the biological and mechanical functions of their native counterparts if they are to provide health benefits to society. However, engineered skin substitute (ESS) only fulfills basic skin functions and fails to match the structural and biophysical characteristics of the human skin, such as missing hair follicle and sweat gland, limiting its use in vivo. Their absences are due to a lack of functioning cell types that instruct human keratinocytes in ESS to make a hair follicle and a lack of an in-depth understanding of essential epithelial-mesenchymal interactions that drive hair follicle formation. In the case of hair follicle engineering, the epithelial-mesenchymal interactions between keratinocytes and their immediate dermal environment need to be precisely modulated to govern hair follicle lineage commitment. The major cell type that constitutes a unique dermal “niche” is a specialized population of fibroblasts, which are located at the base of the hair follicle, called the dermal papilla (DP), and are different from normal human dermal fibroblasts. However, it is of great difficulty to isolate and expand human DP fibroblasts in vitro while maintaining their inductive capacity for tissue engineering purposes. The long-term goal is to develop novel bioengineering approaches to produce a fully functional human skin equivalent with normal microanatomy. The central hypothesis is that hair follicle induction is an emergent property of skin constructs, which requires the interplay of multiple signals and cell types in an inductive microenvironment. The objectives are to systemically explore how to create an inductive microenvironment in ESS to induce hair follicle formation. To achieve this, we have devised a three-pronged strategy addressing hair follicle bioengineering in ESS: 1) mimic the original niche by fabricating composite keratinocyte-DP cell spheroids in micropatterned ESS; 2) enhance intercellular interactions by adding BMP6 and 3) drive the DP phenotype by re-activating master transcription factors. In the first aim, we will determine if multi- cell type spheroids combined with premade hair canals can mimic a natural niche in ESS. We have developed two 3D composite spheroid models and will determine whether they will allow DP fibroblasts to expand in vitro while maintaining DP inductivity to induce hair follicles in a laser micropatterned skin substitute model. In the second aim, we will determine the inductive functions of Bmp6 in stimulating hair follicle formation in ESS. We will dissect the roles of Bmp6 in hair follicle formation and growth. In the third aim, we will determine whether human DP fibroblasts can be reprogrammed to reestablish hair inductivity. We will assess whether in vitro genetic reprogramming of human DP fibroblasts by CRISPRa-mediated expression of master transcription factors promote the ability of composite keratinocyte-DP cell spheroids to induce hair follicles in ESS. This work will provide novel mechanistic knowledge of spatial niche arrangement, inductive signals, and genetic programs that is critical to promote hair follicle neogenesis in ESS. The outcomes are significant because it will accelerate hair follicle bioengineering and advance complex in vitro and in vivo tissue engineering and regenerative medicine.