Stem Cell Based Therapy for Regenerative Endodontics

基于干细胞的再生牙髓治疗

• 批准号: 7666150
• 负责人: GEORGE T.J HUANG
• 金额: --
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7666150
• 关键词: Adult; Aftercare; Amputation; Animals; Apical; Autologous; Back; Bone Marrow; Bone Regeneration; Cell Separation; Cell Therapy; Cells; Cellularity; Classification; Clinical; Clinical Treatment; Clinical Trials; Data; Dental; Dental Pulp; Dental crowns; Dentin; Endodontically-Treated Tooth; Endodontics; Environment; Excision; Failure; Family suidae; Flow Cytometry; Foundations; Future; Gene Expression; Gene Expression Profile; Goals; Growth Factor; Gutta-Percha; Harvest; Health; Histology; Human; Immunohistochemistry; Implant; In Vitro; Light; Mesenchymal Stem Cells; Methods; Miniature Swine; Modality; Modeling; Molecular Profiling; Mus; Natural regeneration; Odontoblasts; Organ; Outcome; Plant Roots; Plants; Procedures; Process; Production; Property; Protocols documentation; Pulp Canals; Pulp Chambers; Research; Reverse Transcriptase Polymerase Chain Reaction; Root Canal Therapy; Rubber; SCID Mice; Services; Simulate; Source; Stem cells; Structure; Study models; System; Technology; Testing; Therapeutic; Time; Tissue Engineering; Tissues; Tooth structure; United States; Vascularization; Western Blotting; Work; angiogenesis; base; cell growth; clinical application; cost; craniofacial; craniofacial repair; deciduous tooth; human stem cells; in vivo; injured; nerve supply; organ regeneration; prospective; public health relevance; regenerative; repaired; research study; scaffold; stem cell therapy; subcutaneous; success; tissue regeneration; tongue papilla

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.

描述（由申请人提供）：该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要。在美国，每年约有 1600 万颗牙齿接受根管治疗（约 140 亿美元），该手术包括去除整个牙髓并用古塔胶（类似橡胶的材料）替换。除了完全截肢外，没有其他有效的方法来修复感染或受伤的牙髓组织。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。去除整个牙髓会带来两个临床问题：1）通常需要进一步的牙科治疗，例如牙冠和桩，这会削弱牙齿的强度； 2）失活的未成熟牙齿脆弱，牙本质结构很少。因此，牙根再生牙髓/牙本质的治疗能力将在临床上有益。我们的初步数据表明，利用干细胞在体外构建的人牙髓样结构并植入人牙齿碎片的牙根管中，可以在SCID小鼠的皮下空间中血管化。因此，我们假设使用干细胞在支架系统的辅助下可以在牙齿中再生牙髓样组织。在本提案中，我们将表征用于牙髓/牙本质再生的牙科干细胞，并分别使用小鼠和小型猪作为异位和原位动物研究模型。有三个具体目标逐步检验假设以达到目标： 目标 1- 表征用于牙髓再生的各种来源的牙髓干细胞。该目标将表征用于牙髓组织再生的各种来源的干细胞。将通过检查与成骨/成牙功效相关的干细胞特性和基因表达谱来分离和表征人牙髓干细胞（hDPSC）、人根尖乳头干细胞（hSCAP）和人脱落乳牙（SHED）干细胞使用体外和体内实验。程序包括流式细胞术、实时RT-PCR、蛋白质印迹、免疫组织化学分析等。目标2-确定SCID小鼠体内人牙髓样组织的血管生成。血管生成的程度对于牙髓再生的成功至关重要。将干细胞接种到含有血管生成生长因子的 D,L-丙交酯和乙交酯 (PLG) 支架中，将其插入人类牙齿碎片的根管间隙中，并皮下植入 SCID 小鼠体内。然后收获植入的牙齿，用于细胞结构、基质产生、成牙本质细胞样细胞的形成，特别是血管结构的组织学分析。将使用免疫组织化学分析再生组织的基因表达谱。目标 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感染并消毒的牙髓空间中进行牙髓再生。来自猪牙齿的牙髓干细胞将在体外种植在支架中，然后种植回消毒的无髓根管空间中进行从头再生牙髓或种植到髓室空间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干细胞疗法临床牙髓学的潜在突破，并为其他器官再生提供范例。 公共健康相关性：在美国，每年约有 1600 万颗牙齿接受根管治疗（约 140 亿美元），该手术需要去除整个牙髓组织，然后用古塔胶（橡胶状材料）代替。这种牙髓组织对于牙齿的整个功能和健康非常重要，去除牙髓组织会使牙齿变弱并且容易出现功能障碍。 除了完全截肢外，没有其他有效的方法来修复感染或受伤的牙髓/牙本质组织。该提案的目的是利用并进一步表征成体牙干细胞用于牙髓/牙本质组织的再生。本提案将利用动物研究模型建立并测试一种利用成体牙干细胞再生牙髓的方法。如果成功，未来可能会进行后续的人体试验，其成功将有望减少对传统侵袭性根管治疗的需求。