Notch signaling and bone fracture healing

描述(由申请人提供)：颅面部和长骨缺损通常表现为愈合延迟或不愈合。虽然自体骨移植和骨形态发生蛋白被广泛用于治疗这种严重的创伤，但这些治疗方法都有局限性。因此，临床上一直需要开发新的方法来促进骨再生。Notch信号通路调节骨前体细胞的增殖和成骨细胞的分化。此外，Notch信号在许多组织中是伤口愈合的重要组成部分，损伤后Notch信号的上调被证明可以促进这些组织的再生。因此，本研究的长期目标是表明Notch信号也调节颅面和长骨组织的再生，并且通过临床适用的组织工程策略上调Notch信号可以促进颅面骨再生。以下三个具体目标是对NIDCR 2009-2013年战略计划(草案)目标1-5的补充，目标1-5是“通过生物、生物工程和生物材料研究方法促进病变或受损的口腔和颅面组织和器官的重建和再生。”对于第一个特定目标，我们将描述颅骨缺损和胫骨骨折愈合过程中Notch信号的特征。Notch报告小鼠将经历直径3毫米的颅骨缺损或双侧胫骨骨折。分别于伤前、伤后1、2、5、10、15、20、40d处死小鼠，进行定量聚合酶链式反应和免疫组织化学分析。在特定的目标2中，我们将确定Notch信号对于成功的颅骨和胫骨再生的关键重要性。具有Cre调节的规范缺口抑制物dnMAML表达的转基因小鼠将经历3 mm的颅骨缺损或双侧胫骨骨折，而Adeno-Cre将局部注射tp永久抑制Notch信号(Adeno-Lacz对照)。分别于伤后5、10、20、40天处死小鼠，进行JCT、组织学和生物力学检测。对于最终的特定目标，我们将开发一种Notch组织工程疗法来促进颅骨再生。作为概念验证，我们将对野生型小鼠的3 mm颅骨缺陷实施Adeno-NICD(Adeno-Lacz对照)，以瞬时上调Notch。然后，我们将Notch Ligand，Jagge-1吸附到一种骨传导生物材料上，并将这些组织工程构建物植入3 mm的颅骨缺损处。本研究的目的是1)研究Notch信号通路在颅面骨愈合中的作用(S)，2)证明Notch手法在临床上可以促进颅面骨再生。 公共卫生相关性：颅面部和长骨缺损通常表现为愈合延迟或不愈合。虽然自体骨移植和骨形态发生蛋白被广泛用于治疗这种严重的创伤，但这些治疗方法都有局限性。因此，临床上一直需要开发新的方法来促进骨再生。本研究的目的是1)研究Notch信号通路在颅面骨愈合中的作用(S)，2)证明Notch手法在临床上可以促进颅面骨再生。

DESCRIPTION (provided by applicant): Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The Notch signaling pathway regulates osteoprogentor proliferation and osteoblast differentiation. Additionally, Notch signaling is an essential component for wound healing in many tissues, and Notch upregulation following injury has been shown to enhance regeneration of those tissues. Therefore, the long-term objectives of this study are to show that Notch signaling also regulates regeneration of craniofacial and long bone tissue, and that upregulation of Notch signaling through a clinically-applicable tissue engineering strategy can enhance craniofacial bone regeneration. The following three specific aims complement the NIDCR's strategic plan for 2009-2013 (draft) objective 1-5, which is to 'Facilitate reconstruction and regeneration of diseased or damaged oral and craniofacial tissues and organs through biological, bioengineering and biomaterials research approaches.' For the first specific aim we will characterize Notch signaling during calvarial defect and tibial bone fracture healing. Notch reporter mice will undergo a 3 mm diameter calvarial defect or bilateral tibial fractures. Mice will be sacrificed pre-injury and at 1, 2, 5, 10, 15, 20 and 40 days post-injury for qPCR and immunohistochemistry analysis. In specific aim 2, we will determine the critical importance of Notch signaling for successful calvarial and tibial bone regeneration. Transgenic mice with Cre-regulated expression of the canonical notch inhibitor dnMAML will undergo a 3 mm calvarial defect or bilateral tibial fractures and adeno-Cre will be locally administered tp permanently inhibit Notch signaling (adeno-LacZ control). Mice will be sacrificed at 5, 10, 20 and 40 days post-injury for |JCT, histology, and biomechanical testing. For the final specific aim we will develop a Notch tissue engineering therapy to enhance calvarial bone regeneration. As a proof of concept, we will administer adeno-NICD (adeno-LacZ control) to 3 mm calvarial defects in wild type mice to transiently upregulate Notch. We will then adsorb Notch ligand, Jagged-1 onto an osteoconductive biomaterial and implant these tissue engineered constructs in 3 mm calvarial defects. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration. Public Health Relevance: Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration.