Identifying Approaches to Enhance Bone and Cartilage Regeneration

骨折愈合不好会导致严重的发病率和死亡率。此外， 需要适当的骨形成对于从关节置换到手术的成功结果至关重要 脊柱融合到牙科植入物。在老龄化的人口中，生物医学的需求甚至更大。最近的 由8个亚群组成的能够形成全部3个亚群的骨祖细胞的鉴定 骨骼、骨骼、软骨和基质的组成部分，有望对改善 这些疗法的结果。仍然存在的一个关键问题是，影响细胞最终命运的因素是什么 这些骨骼祖细胞。我们关于基质细胞蛋白DEL1的数据表明，它对 骨骼修复。我们有初步数据表明，它对骨骼祖细胞生物学有直接影响。 我们认为DEL1在它们的生物学中起着重要的作用。 在此之前，我们的数据显示DEL1对软骨细胞有生物学效应，但对成骨细胞没有影响。我们 显示小鼠骨骼干细胞(MSSCs)以及骨、软骨、基质的数量减少 祖细胞(BCSPs)，在骨折愈合中最重要的骨骼祖细胞，我们 提出骨折后骨形成减少的机制是由于对骨骼祖细胞的影响 骨折后的伸展能力。我们计划首先通过将mSSCs和BCSPs从基因敲除中分离出来来研究这一点 (KO)和野生型(WT)小鼠，比较它们的生物学特性。我们将利用体外实验检测BCSPs的生物学特性 细胞增殖、成骨和成软骨能力的检测。BCSP产生不同的表型 称为骨折后具有较大增殖能力和成骨潜力的骨折BCSP(f-BCSP)， 我们将测试KO小鼠是否会发生同样的变化。我们将进行体内分析，以确定 通过将BCSP植入肾被膜以检查它们形成骨的情况来成骨。 要了解DEL1如何影响骨折后的骨形成，关键问题是了解 WT和KO小鼠骨折后骨骼祖细胞发生的变化。我们将使用世系跟踪技术 目前在我们实验室中的小鼠正在研究骨骼的单个克隆的子细胞的命运 祖细胞。 最后一系列实验代表了我们将DEL1的用法转化为潜在用法的初步方法 治疗。首先，我们将进行仔细控制的微阵列实验，以确定KO中改变的途径 骨骼祖细胞与WT相比。我们将检测外源DEL1促进活性和 体外培养的骨骼干细胞的生长情况。我们将测试DEL1刺激骨骼和软骨的能力 移植到肾被膜内的骨祖细胞的形成。最后，我们将检查是否 外源性DEL1蛋白置入骨折部位可促进正常骨折愈合。我们还将使用 用后肢照射小鼠建立骨折愈合不良的单独模型。 综上所述，我们将研究Del1如何影响骨骼祖细胞的生物学。除了……之外 提供可能将这些发现转化为疗法的步骤，这些实验可以为我们提供 关于缺乏Del1如何导致骨折愈合中骨骼减少的机械答案。他们将提供 洞察影响骨骼祖细胞潜在命运的因素。

Failure to properly heal bone fractures is associated with significant morbidity and mortality. Additionally, the need to properly form bone is vital to successful outcomes from surgeries ranging from joint replacement to spinal fusion to dental implants. In an aging population, the biomedical need is even greater. The recent identification of skeletal progenitor cells, composed of 8 subpopulations, capable of forming all three components of the skeleton, bone, cartilage and stroma, promises to have a major impact on improving the outcomes for these therapies. A key question that remains is what are the factors affecting eventual cell fate of these skeletal progenitor cells. Our data on the matricellular protein DEL1 have shown it has an impact on skeletal repair. We have preliminary data indicating that it has a direct effect on skeletal progenitor cell biology and we propose that DEL1 plays an important role in their biology. Prior to this, our data had shown DEL1 had a biological effect on chondrocytes, but not on osteoblasts. We demonstrated decreased numbers of mouse skeletal stem cells (mSSCs), and bone, cartilage, stroma progenitors (BCSPs), the skeletal progenitor most important in fracture healing in the fracture callus, and we propose the mechanism for decreased bone formation after fracture is due to an effect on skeletal progenitors ability to expand after fracture. We plan on examining this first by isolating mSSCs and BCSPs, from knockout (KO) and wild type (WT) mice and comparing their biology. We will examine the biology of BCSPs using in vitro assays for cell proliferation, osteogenic and chondrogenic potential. BCSPs develop a different phenotype termed the fracture BCSP (f-BCSP) that has greater proliferative ability and osteogenic potential after fracture, and we will test whether the same change occurs in our KO mice. We will perform in vivo assays for osteogenesis by implanting BCSPs into the renal capsule to examine how well they form bone. A key question in understanding how DEL1 affects formation of bone after fracture is to understand what happens to skeletal progenitors after fracture in WT and KO mice. We will use lineage tracing techniques with mice currently in our laboratory to examine the fate of daughter cells from individual clones of skeletal progenitors cells. The final series of experiments represent our initial approaches to translating use of DEL1 into potential therapies. First, we will perform a carefully controlled microarray experiment to identify pathways altered in KO skeletal progenitors compared to WT. We will examine the ability of exogenous DEL1 to promote viability and growth of skeletal stem cells grown in vitro. We will test the ability of DEL1 to stimulate bone and cartilage formation of skeletal progenitor cells implanted into the renal capsule. Finally, we will examine whether exogenous DEL1 protein placed at a fracture site can enhance normal fracture healing. We will also use a separate model of poor fracture healing with mice that have undergone hindlimb irradiation. In summary, we will examine how Del1 affects the biology of skeletal progenitor cells. In addition to providing steps to potentially translate these discoveries into therapies, these experiments can provide us with mechanistic answers about how lack of Del1 leads to decreased bone in fracture healing. They will provide insight into the factors that affect the potential fate of skeletal progenitor cells.