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

工程皮肤组织必须复制其天然对应物的生物和机械功能， 为社会提供健康福利。然而，工程皮肤替代品（ESS）只能满足基础皮肤 功能和不匹配的人体皮肤的结构和生物物理特性，如缺失 毛囊和汗腺，限制了其在体内的使用。他们的缺席是由于缺乏功能细胞类型 指导ESS中的人类角质形成细胞形成毛囊， 驱动毛囊形成的重要上皮-间充质相互作用。在毛囊的情况下 工程，角质形成细胞和它们的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谱系定型。主要的细胞类型， 构成一个独特的真皮“生态位”是一个专门的成纤维细胞群，位于基底部， 毛囊称为真皮乳头（DP），与正常人真皮成纤维细胞不同。然而，在这方面， 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， 用于组织工程的目的。长期目标是开发新的生物工程方法， 一种功能齐全的人体皮肤，具有正常的显微解剖结构。中心假设是毛囊 诱导是皮肤结构的一种新特性，它需要多种信号和细胞的相互作用。 在一个微环境中进行归纳。其目标是系统地探讨如何建立一个归纳 ESS微环境诱导毛囊形成。为此，我们制定了一个三管齐下的 ESS中毛囊生物工程的解决策略：1）通过制造复合材料来模拟原始生态位 角化细胞-DP细胞球状体在微图案化ESS中; 2）通过添加BMP 6和 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，我们将确定是否多- 细胞型球状体与预制的头发管道相结合，可以模仿ESS中的自然生态位。 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增 同时保持DP诱导性以在激光微图案化皮肤替代物模型中诱导毛囊。在 第二个目的是确定Bmp 6在ESS中刺激毛囊形成的诱导功能。 将剖析Bmp 6在毛囊形成和生长中的作用。在第三个目标中，我们将确定 人DP成纤维细胞可被重编程以重建毛发诱导性。我们将评估是否在体外遗传 通过CRISPRa介导的主转录因子表达重编程人DP成纤维细胞 促进复合角质形成细胞-DP细胞球体诱导ESS中毛囊的能力。这项工作将 提供了空间生态位排列，诱导信号和遗传程序的新机制知识， 在ESS中促进毛囊新生是至关重要的。结果是显著的，因为它会加速头发的生长。 卵泡生物工程和推进复合体的体内外组织工程和再生医学。

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.