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，从而减少对根管治疗的需求。在美国，每年大约有1600万颗牙齿接受根管治疗（约140亿美元）——这一过程包括去除整个牙髓，并用胶状材料代替。除了完全截肢外，没有其他有效的方法来修复感染或损伤的牙髓组织。近年来，牙髓干细胞分离和组织工程技术的发展为牙髓组织再生提供了新的可能性。移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，如冠和桩，这会削弱牙齿；2)失活的未成熟牙脆弱，牙本质结构小。因此，再生根髓/牙本质的治疗能力在临床上是有益的。我们的初步数据表明，利用干细胞体外构建人牙髓样结构并植入人牙碎片管中，可以在SCID小鼠皮下空间血管化。因此，我们假设牙髓样组织可以用干细胞辅助支架系统再生。在本研究中，我们将对牙髓/牙本质再生的牙干细胞进行表征，并分别以小鼠和小型猪作为异位和正位动物研究模型。有三个具体的目标，逐步测试假设，以达到目标：目标1-表征牙髓再生的各种来源的牙髓干细胞。这一目标将描述用于牙髓组织再生的各种来源的潜在干细胞。人类牙髓干细胞（hDPSCs）、人类根尖乳头干细胞（hSCAP）和人类脱落乳牙干细胞（SHED）将被分离出来，并通过体外和体内实验检测其干细胞特性和与成骨/成牙潜能相关的基因表达谱。方法包括流式细胞术、实时RT-PCR、Western blot、免疫组织化学分析等。目的2-测定SCID小鼠体内人髓样组织血管生成情况。血管生成的程度是牙髓再生成功的关键。将干细胞植入含有血管生成生长因子的D， l -丙交酯和甘油酯（PLG）支架中，植入人牙碎片根管间隙，皮下植入SCID小鼠。移植后的牙齿将被采集，用于细胞结构、基质生成、成牙细胞样细胞的形成，特别是血管结构的组织学分析。用免疫组化方法分析再生组织的基因表达谱。目的3-在猪的相关组织环境中测试牙髓再生。本实验猪模型模拟了人类临床应用，利用自体干细胞在感染后消毒的髓腔内进行髓再生。将猪牙髓干细胞植入体外支架中，植入消毒后的无髓根管空间进行髓体再生或植入髓室空间进行部分髓体再生。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. 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.