Biomimetic Inductive Scaffolds for Tooth Organ Engineering

描述（由申请人提供）：本修订提案的总体目标是设计和制造模仿某些胚胎组织如何诱导胚胎中整个器官形成的仿生支架，并最终使用这些发育启发材料在成年动物中设计人工组织和器官。作为一种原理证明，我们建议制造多功能支架，诱导成体间充质干细胞（aMSCs）和成体口腔上皮细胞（aOECs）形成分化的牙齿。我们的方法是基于我们过去的工作，这些工作表明，在胚胎发生过程中，牙上皮通过刺激间质凝聚反应将其诱导能力转移到未分化的牙间质，并且由此产生的细胞物理压实足以触发这种体外发育开关以及随后的体内牙齿分化。此外，我们现在包括新的初步结果，表明我们可以制造热敏聚合物支架，在体温下收缩并人工诱导间充质凝结，并且可以通过体外加热和将这些材料与胚胎牙间充质细胞结合并将其植入小鼠体内的肾包膜下来刺激牙齿分化。基于对胚胎牙诱导过程的分析，我们认为在这些支架中加入其他有助于正常牙齿发育的关键细胞外基质（ECM）分子和形态因子可以提高人工牙诱导过程的效率；我们还计划优化机械感应支架的设计和制造。因此，本研究的长期目标是制备仿生聚合物支架，通过对aMSCs进行重编程，使其分化为诱导间质，从而指导正常aoec在体内形成分化的牙齿。本研究的具体目标包括：1)制造一种机械可驱动的仿生聚合物支架，通过产生细胞压实诱导aMSCs在体外进行牙源性分化；2)鉴定胚胎发生过程中介导牙齿形成的分子，从而增强压实诱导的体外牙齿分化；3)在小鼠模型中使用优化的仿生支架与aMSCs和aOECs结合，在体内生物工程分化牙齿。

DESCRIPTION (provided by applicant): The overall goal of this REVISED proposal is to design and fabricate biomimetic scaffolds that mimic how certain embryonic tissues induce whole organ formation in the embryo, and ultimately, to use these developmentally-inspired materials to engineer artificial tissues and organs in adult animals. As a proof-of- principle, we propose to fabricate multifunctional scaffolds that induce formation of differentiated tooth starting with adult mesenchymal stem cells (aMSCs) and adult oral epithelial cells (aOECs). Our approach is based our past work which shows that during embryogenesis, the dental epithelium transfers its inductive capabilities to undifferentiated dental mesenchyme by stimulating a mesenchymal condensation response, and that the resulting physical compaction of cells is sufficient to trigger this developmental switch in vitro as well as subsequent tooth differentiatio in vivo. In addition, we now include new preliminary results that show we can fabricate thermosensitive polymer scaffolds that shrink and artificially induce mesenchymal condensation when placed at body temperature, and that tooth differentiation can be stimulated both by warming in vitro and by combining these materials with embryonic dental mesenchymal cells and implanting them under the kidney capsule in mice in vivo. Based on analysis of the embryonic tooth induction process, we believe that we can enhance the efficiency of this artificial induction process by including other key extracellular matrix (ECM) molecules and morphogens in these scaffolds that contribute to normal tooth development; we also plan to optimize the design and fabrication of the mechano-inductive scaffolds. Thus, the long-term goal of this proposal is to fabricate biomimetic polymer scaffolds that can reprogram aMSCs to differentiate into inductive mesenchyme that instructs normal aOECs to form a differentiated tooth in vivo. The Specific Aims of this proposal include: 1) to fabricate a mechanically actuatable biomimetic polymer scaffold that induces aMSCs to undergo odontogenic differentiation in vitro by producing cell compaction, 2) to identify molecules that mediate tooth formation during embryogenesis that can enhance compaction-induced tooth differentiation in vitro, and 3) to bioengineer a differentiated tooth in vivo using an optimized biomimetic scaffold in combination with aMSCs and aOECs in a mouse model.