Stem Cell Based Therapy for Regenerative Endodontics

描述(由申请人提供)：这项建议的长期目标是再生和修复牙髓/牙本质，从而减少牙髓治疗程序的需要。在美国，每年约有1600万颗牙齿接受根管治疗(约140亿美元)--这是一种去除整个牙髓并用杜仲胶(橡胶状材料)取代的过程。除完全截肢外，目前尚无其他有效的方法修复感染或损伤的牙髓组织。牙髓干细胞分离和组织工程技术的最新进展揭示了牙髓组织再生的可能性。去除整个牙髓带来两个临床问题：1)最常需要进一步的牙科工作，如牙冠和牙桩，以削弱牙齿；2)失活的未成熟牙齿脆弱，牙本质结构很少。因此，再生牙髓/牙本质的治疗能力在临床上是有益的。我们的初步数据显示，使用干细胞体外构建的人牙髓样结构植入人类牙齿碎片的根管中，可以在SCID小鼠的皮下空间形成血管。因此，我们假设牙髓样组织可以使用干细胞在支架系统的辅助下在牙齿中再生。在这个方案中，我们将表征牙髓/牙本质再生的牙科干细胞，并分别以小鼠和小型猪作为异位和原位动物研究模型。有三个特定的目标来逐步检验假设以达到目标：目标1-表征不同来源的牙髓干细胞用于牙髓再生。这一目标将表征用于牙髓组织再生的各种来源的未来干细胞。从人牙髓干细胞(HDPSCs)、根尖乳头干细胞(HSCAP)和人脱落乳牙干细胞(SHEAD)中分离并鉴定它们的干细胞特性和与成骨/成牙潜能相关的基因表达谱。程序包括流式细胞术、实时定量RT-PCR、Western印迹、免疫组织化学分析等。目的2-检测SCID小鼠体内人牙髓样组织的血管生成。血管生成的程度对牙髓再生的成功与否至关重要。将种植在含有血管生长因子的D，L-丙交酯和乙交酯支架中的干细胞植入人牙齿碎片的根管间隙，并植入SCID小鼠皮下。然后，将采集植入的牙齿进行细胞学分析、基质产生、成牙本质细胞样细胞的形成，特别是血管。再生组织的基因表达谱将用免疫组织化学方法进行分析。目的3-在相关的组织环境中测试猪的牙髓再生。这个实验猪模型模拟了人类的实际临床应用，使用自体干细胞在被感染的牙髓间隙再生牙髓，然后进行消毒。将猪牙的牙髓干细胞接种于体外支架内，种植回消毒后的无髓根管腔内进行牙髓再生，或种植于牙髓室腔内进行部分牙髓再生。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着使用干细胞疗法在临床牙髓治疗方面的潜在突破，并为其他器官再生提供范例。 与公共卫生相关：在美国，每年约有1600万颗牙齿接受根管治疗(约140亿美元)-这一过程需要移除整个牙髓组织，然后用杜仲胶(橡胶状材料)取代。这种牙髓组织对牙齿的整体功能和健康很重要，去除它会使牙齿变得虚弱，容易受到功能障碍的影响。除了完全截肢外，目前还没有其他有效的方法来修复感染或损伤的牙髓/牙本质组织。这项建议的目的是利用成人牙干细胞来再生牙髓/牙本质组织，并对其进行进一步鉴定。利用动物研究模型，将建立一种方法，并在该提案中测试使用成人牙科干细胞再生牙髓。如果成功，未来可能会进行后续的人体试验，它的成功将保证减少对传统侵入性根管治疗的需求。

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. PUBLIC HEALTH RELEVANCE: About 16 million teeth receive root canal treatment (~$14 billion) per year in the United States- a procedure that requires the removal of the entire dental pulp tissue which is then replaced with gutta percha (rubber-like material). This pulp tissue is important for the entire function and the health of the tooth, removing which renders the tooth weakened and susceptible to functional failures. There have been no other effective ways to repair the infected or injured pulp/dentin tissue besides complete amputation. The purpose of this proposal is to utilize and further characterize the adult dental stem cells for the regeneration of pulp/dentin tissues. Using animal study models, a method will be established and tested in this proposal to regenerate dental pulps using adult dental stem cells. If successful, a subsequent human trial may be carried out in the future and its success will promise a reduced need of traditional aggressive root canal therapies.