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

描述(申请人提供)：Nell-1的体外表达显著增加了成骨细胞的分化和矿化。在转基因小鼠中过表达Nell-1显著增加了颅骨的形成。Nell-1和BMP2的比较在啮齿类颅骨缺损模型中显示出相似的骨诱导特性。此外，Nell-1与骨形态发生蛋白(BMP2)在诱导碱性磷酸酶活性方面显示出潜在的相加或协同作用。总体而言，我们假设Nell-1可以促进颅骨再生，并可能增强BMP介导的骨再生。为了检验这一假设，我们提出了两个目标。 目的1分别优化Nell-1、BMP2和BMP7对骨形成所需的浓度(在大鼠颅骨临界大小缺损模型中)，然后确定优化后的Nell-1、BMP2或BMP7对特定细胞分化状态(即增殖和凋亡)和骨标记基因表达的影响。在目标1A中，我们的工作假设是优化骨形成的Nell-1、BMP2或BMP7的浓度会有所不同。每种因子的最佳单一治疗剂量(DoseOPT)将作为Aim 2B中提出的体内组合的上限。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BMPs的生物响应性。由此推论，Nell-1和BMPs可能通过不同的途径诱导成骨，这些途径涉及不同的细胞表型/分化状态。为了评估这一点，我们将在组织学上将成骨分化标记物的表达与增殖和凋亡的细胞表型共存。 目标2将在体外探索Nell-1和BMP组合制剂对特定参数如增殖、成骨细胞标志物表达、细胞凋亡和矿化能力的影响，然后尝试使用与目标1相同的颅骨模型在体内翻译这些配方。对于目标2A，我们的工作假设是，特定的Nell-1/BMP2或Nell-1/BMP7组合比例在体外将比单独使用Nell-1、BMP2或BMP7诱导更多的矿化。Aim 2A中优化的Nell-1/BMP组合将作为Aim 2B中剂量的建议Nell-1：BMP比例。对于Aim 2B，我们的工作假设是，体外优化的Nell-1：BMP比率可以与来自Aim 1的体内优化的DoseOPT数据相结合，以更好地预测Aim 2B的最佳体内Nell-1/BMP剂量需求。来自AIMS 1B/2a2的细胞和分子分析数据将进一步微调AIM 2B中Nell-1/BMP的剂量。

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