Stem Cell Based Therapy for Regenerative Endodontics

描述（由申请人提供）：本提案的长期目标是再生和修复牙髓/牙本质，从而减少牙髓手术的需求。在美国，每年约有1600万颗牙齿接受根管治疗（约140亿美元），这是一种包括去除整个牙髓并用古塔胶（橡胶状材料）替代的手术。对于感染或损伤的牙髓组织，除完全切断外，尚无其他有效的修复方法。牙髓干细胞分离和组织工程技术的最新进展为牙髓组织再生的可能性提供了新的思路。去除整个牙髓会带来两个临床问题：1）通常需要进一步的牙科工作，如削弱牙齿的牙冠和桩; 2）失活的未成熟牙齿很脆弱，几乎没有牙本质结构。因此，再生牙根的牙髓/牙本质的治疗能力将是临床有益的。我们的初步数据表明，在体外使用干细胞构建的人牙髓样结构，并植入人牙碎片的根管内，可以在SCID小鼠的皮下空间血管化。因此，我们假设牙髓样组织可以再生牙齿使用干细胞辅助支架系统。在这个建议中，我们将表征牙髓/牙本质再生的牙齿干细胞，并分别使用小鼠和小型猪作为异位和原位动物研究模型。有三个具体的目标，逐步测试的假设，以达到目标：目的1-表征牙髓再生的各种来源的牙髓干细胞。这一目标将表征用于牙髓组织再生的各种来源的有前景的干细胞。将分离人牙髓干细胞（hDPSC）、来自根尖乳头的人干细胞（hSCAP）和来自人脱落乳牙（SHED）的干细胞，并通过使用体外和体内实验检查其干细胞特性和与成骨/成牙潜能相关的基因表达谱来表征。方法包括流式细胞术、实时定量RT-PCR、Western blot、免疫组化等。目的2-检测人牙髓样组织在SCID小鼠体内的血管生成情况。血管生成的程度对于牙髓再生的成功至关重要。将干细胞接种在含有血管生成生长因子的D，L-丙交酯和乙交酯（PLG）支架中，将其插入人牙齿碎片的根管间隙中，并皮下植入SCID小鼠中。然后将收获植入的牙齿用于细胞构成、基质产生、成牙本质细胞样细胞的形成以及特别是血管分布的组织学分析。再生组织的基因表达谱将用免疫组织化学分析。目的3-在猪的相关组织环境中测试牙髓再生。该实验猪模型模拟了人类的实际临床应用，使用自体干细胞在已经感染然后消毒的牙髓空间中进行牙髓再生。将来自猪牙齿的牙髓干细胞接种在体外支架中，并将其种植回消毒的无髓根管间隙中进行重新牙髓再生或种植到髓室间隙中进行部分牙髓再生。再生组织的血管化和神经支配将通过组织学和免疫组织化学来确定。该项目的成功将代表临床牙髓病学使用干细胞治疗的潜在突破，并为其他器官再生提供范例。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to regenerate and repair dental pulp/dentin thereby reducing the need of endodontic procedures. Approximately 16 million teeth receive root canal treatment (~$14 billion) per year in the US-a procedure consisting of the removal of the entire pulp and replacing it with gutta percha (rubber-like material). There have been no other effective ways to repair the infected or injured pulp tissue besides complete amputation. Recent advances in pulp stem cell isolation and tissue engineering technologies have shed light on the possibility of pulp tissue regeneration. Removing the entire pulp poses two clinical problems: 1) further dental work such as crown and post that weaken the tooth are most often required; 2) devitalized immature teeth are weak with little dentin structure. Therefore the therapeutic capacity to regenerate pulp/dentin of the root would be clinically beneficial. Our preliminary data showed that human pulp-like structure constructed in vitro using stem cells and implanted in canals of human tooth fragments can be vascularized in the subcutaneous space of SCID mice. Therefore, we hypothesize that pulp-like tissue can be regenerated in tooth using stem cells aided with a scaffold system. In this proposal, we will characterize dental stem cells for regeneration of pulp/dentin and use mice and minipigs as ectopic and orthotopic animal study models respectively. There are three specific aims that stepwisely test the hypothesis to reach the goal: Aim 1- To characterize pulp stem cells of various sources for pulp regeneration. This aim will characterize prospective stem cells of various sources for pulp tissue regeneration. Human dental pulp stem cells (hDPSCs), human stem cells from apical papilla (hSCAP) and stem cells from human exfoliated deciduous teeth (SHED) will be isolated and characterized by examining their stem cell properties and gene expression profiles relevant to osteo/odontogenic potency using in vitro and in vivo experimentation. Procedures include flow cytometry, real-time RT-PCR, Western blot, immunohistochemical analysis, etc. Aim 2- To determine angiogenesis of human pulp-like tissues in vivo in SCID mice. The extent of angiogenesis is critical to the success of pulp regeneration. Stem cells seeded in D,L-lactide and glycolide (PLG) scaffolds containing angiogenic growth factors will be inserted into root canal space of human tooth fragments and implanted subcutaneously into SCID mice. The implanted teeth will then be harvested for histological analysis of cellularity, matrix production, formation of odontoblast-like cells and particularly vascularity. Gene expression profile of the regenerated tissue will be analyzed with and immunohistochemistry. Aim 3- To test pulp regeneration in a relevant tissue environment in swine. This experimental swine model simulates actual clinical applications in humans using autologous stem cells for pulp regeneration in pulp space that has been infected and then disinfected. Pulp stem cells from swine teeth will be seeded in scaffolds in vitro and planted back to the disinfected pulpless root canal space for de novo pulp regeneration or planted into pulp chamber space for partial pulp regeneration. The vascularization and innervations of the regenerated tissue will be determined with histology and immunohistochemistry. The success of this project would represent a potential breakthrough in clinical endodontics using stem cell therapy and serve as an example for other organ regeneration.