COMPARATIVE ANALYSIS OF Nell-1 vs BMPs IN CALVARIAL BONE REGENERATION

描述（由申请人提供）：体外Nell-1的表达显著增加成骨细胞分化和矿化。在转基因小鼠中Nell-1的过表达显著增加了颅骨骨形成。Nell-1与BMP 2的比较在啮齿动物颅骨缺损模型中证明了相当的骨诱导性质。此外，Nell-1与骨形态发生蛋白（BMP 2）在诱导碱性磷酸酶活性方面表现出潜在的相加或协同作用。总之，我们假设Nell-1可以增强颅骨骨再生，并可能增强BMP介导的骨再生。为了验证这个假设，我们提出了两个目标。 目的1将分别优化骨形成所需的Nell-1、BMP 2和BMP 7的浓度（在大鼠颅骨临界尺寸缺损模型中），然后确定优化的Nell-1、BMP 2或BMP 7添加对特定细胞分化状态（即，增殖和凋亡）和骨标记基因表达。在目标1A中，我们的工作假设是用于优化骨形成的Nell-1、BMP 2或BMP 7浓度将变化。每个因素的最佳单药治疗剂量（DoseOPT）将作为目标2B中拟定的体内联合给药的体内阈值上限。在目标1B中，我们的工作假设是细胞类型/分化阶段可以显著影响对优化的Nell-1或BMP的生物反应性。通过推论，Nell-1和BMP可以通过涉及细胞表型/分化状态差异的不同途径诱导骨形成。为了评估这一点，我们将在组织学上共同定位成骨细胞分化标志物表达与增殖和凋亡细胞表型。 目的2将探索组合Nell-1和BMP制剂在体外对特定参数如增殖、成骨细胞标志物表达、凋亡和矿化能力的影响，然后尝试使用与目的1相同的颅骨模型在体内翻译这些制剂。对于目标2A，我们的工作假设是，体外特定的Nell-1/BMP 2或Nell-1/BMP 7组合比例将比单独的Nell-1、BMP 2或BMP 7诱导更多的矿化。目标2A中优化的Nell-1/BMP组合将用作目标2B中给药的建议Nell-1：BMP比率。对于目标2B，我们的工作假设是体外优化的Nell-1：BMP比率可以与来自目标1的体内优化的DoseOPT数据组合，以更好地预测目标2B的最佳体内Nell-1/BMP剂量要求。来自目标1B/2A 2的细胞和分子分析数据将进一步微调目标2B中的Nell-1/BMP剂量。

DESCRIPTION (provided by applicant): Expression of Nell-1 in vitro significantly increased osteoblast differentiation and mineralization. Overexpression of Nell-1 in transgenic mice significantly increased calvarial bone formation. Comparison of Nell-1 with BMP2 demonstrated comparable osteoinductive properties in a rodent calvarial defect model. In addition, Nell-1 exhibited a potential additive or synergistic effect with bone morphogenetic proteins (BMP2) on inducing alkaline phosphatase activity. Overall, we hypothesize that Nell-1 can enhance calvarial bone regeneration and perhaps enhance BMP mediated bone regeneration. To test this hypothesis, we have proposed two Aims. Aim 1 will separately optimize the concentrations of Nell-1, BMP2, and BMP7 required for bone formation (in a rat calvarial critical sized defect model) and then determine the effects of optimized Nell-1, BMP2, or BMP7 addition on specific cellular differentiation states (i.e., proliferation and apoptosis) and bone marker gene expression. In Aim 1A, our working hypothesis is Nell-1, BMP2, or BMP7 concentrations for optimized bone formation will vary. The optimal monotherapy dose (DoseOPT) for each factor will serve as an upper in vivo threshold for the in vivo combinations proposed in Aim 2B. In Aim 1B, our working hypothesis is that the cell type/differentiation stage can significantly impact the bioresponsiveness to optimized Nell-1 or BMPs. By corollary, Nell-1 and BMPs may induce bone through distinct pathways that involve differences in cell phenotype/differentiation state. To assess this, we will histologically co-localize osteoblastic differentiation marker expression with proliferative and apoptotic cellular phenotypes. Aim 2 will explore the effects of combinatorial Nell-1 and BMP formulations in vitro on specific parameters such as proliferation, osteoblast marker expression, apoptosis, and mineralization capacity and then attempt to translate these formulations in vivo using the same calvarial model as Aim 1. For Aim 2A, our working hypothesis is that specific Nell-1/BMP2 or Nell-1/BMP7 combination ratios in vitro will induce more mineralization than Nell-1, BMP2, or BMP7 alone. Optimized Nell-1/BMP combinations in Aim 2A will serve as a suggested Nell-1:BMP ratio for dosing in Aim 2B. For Aim 2B, our working hypothesis is that in vitro optimized Nell-1:BMP ratios can be combined with the in vivo optimized DoseOPT data from Aim 1 to better predict optimal in vivo Nell-1/BMP dose requirements for Aim 2B. The cellular and molecular analyses data from Aims 1B/2A2 will further fine tune Nell-1/BMP dosing in Aim 2B.Project Narrative