Coupling osteoinductive factors to graft materials to promote osteoregeneration

描述（由申请人提供）：由于自体骨移植的局限性，同种异体、异种移植物和同种异体材料通常用于骨移植手术。然而，这些材料缺乏成骨性。提出的研究旨在将骨诱导因子偶联到这些替代移植物源的表面，以增强骨缺乏部位的骨再生。我们的研究小组正在利用一种独特的方法，将钙结合氨基酸结构域七谷氨酸（E7）添加到从BMP2衍生的骨诱导肽中。临床前研究表明，在皮下（异位）和下颌缺损模型中，结合e7修饰的BMP2肽（E7-BMP2pep）的移植物材料比未涂覆的材料能显著促进新骨的合成。重要的是，骨形成与全长rBMP2包被移植物诱导的骨形成相当，在某些情况下甚至更大，并且E7-BMP2pep组没有发现副作用。相比之下，植入rBMP2的动物出现了慢性炎症和白内障。我们假设E7-BMP2pep是一种有效、安全且易于翻译的药物，可用于提高各种骨移植材料的性能，包括同种异体移植物、异种移植物和同种异体。本研究的主要目标有两个：(1)全面分析宿主对E7- BMP2pep的反应，以推进翻译研究（目的1）；(2)阐明BMP2模拟肽激活骨祖细胞的机制，更广泛的目标是鉴定具有最大骨诱导潜力的BMP2衍生物。目的1：宿主对E7-BMP2pep偶联移植物材料的反应。采用下颌缺损模型评估E7-BMP2pep偶联材料对免疫和异物反应、纤维化、血管生成、新生骨的数量和质量以及种植体整合的影响。还将监测肽和rBMP2从植入部位外的传播情况。我们假设E7-BMP2pep将紧密定位于缺损内，促进快速有效的骨愈合。利用细胞培养和头颅器官培养系统，研究e7修饰的BMP2模拟肽调控干细胞/祖细胞的机制。此外，将开发具有激活突变的BMP2肽变体，并在补充研究中，将E7结构域设计到全长rBMP2上，以改善与载体的耦合。这些BMP变异将被评估受体结合活性和诱导细胞信号传导和成骨细胞发生。增强的骨诱导变异将被下颌骨种植研究证实。Aim 2的主要目标是确定理想的BMP2载货分子，用于E7定向偶联接枝材料。

DESCRIPTION (provided by applicant): Due to limitations associated with autografted bone, allograft, xenograft and alloplast materials are commonly used for bone grafting procedures. However these materials lack osteoinductivity. The proposed research aims to couple osteoinductive factors to the surface of these alternative graft sources in order to enhance osteoregeneration in sites of bone deficiency. Our group is utilizing a unique approach involving the addition of a calcium-binding amino acid domain, heptaglutamate (E7), to an osteoinductive peptide derived from BMP2. Preclinical studies show that graft materials coupled with the E7-modified BMP2 peptide (E7-BMP2pep) stimulated significantly greater new bone synthesis than uncoated materials in both a subcutaneous (ectopic) and mandibular defect model. Importantly, bone formation was comparable to, and in some cases greater than, that induced by graft coated with full-length rBMP2, and no side effects were noted for the E7-BMP2pep group. In contrast, animals implanted with rBMP2 developed chronic inflammation and cataracts. We hypothesize that the E7-BMP2pep is an effective, safe, and readily translatable agent that can be used to enhance the performance of a wide array of bone grafting materials including allograft, xenograft and alloplast. There are two major objectives of this proposal: (1) to comprehensively analyze host response to the E7- BMP2pep in order to advance the research toward translation (Aim 1); and (2) to elucidate the mechanism by which BMP2 mimetic peptides activate osteoprogenitor cells, with the broader goal of identifying a BMP2 derivative with maximal osteoinductive potential. AIM 1: Host response to E7-BMP2pep-coupled graft materials A mandibular defect model will be used to assess the effects of E7-BMP2pep coupled materials on immune and foreign body response, fibrosis, angiogenesis, quantity and quality of newly-formed bone, and implant integration. Dissemination of the peptides and rBMP2 away from the implant site will also be monitored. We hypothesize that E7-BMP2pep will remain tightly localized within the defect, promoting rapid and effective bone healing. AIM 2: Molecular characterization and engineering of BMP2 mimetics to enhance osteoinductive potential Using cell culture and calvarial organ culture systems, the mechanisms by which E7-modified BMP2 mimetic peptides regulate stem/progenitor cells will be elucidated. Additionally, BMP2 peptide variants with activating mutations will be developed, and in complementary studies, an E7 domain will be engineered onto full-length rBMP2 to improve coupling to carriers. These BMP variants will be evaluated for receptor binding activity and induction of cell signaling and osteoblastogenesis. Enhanced osteoinductivity of the variants will be confirmed by mandibular implant studies. The broad goal of Aim 2 is to identify the ideal BMP2 cargo molecule for E7- directed coupling to graft materials.