Direct and indirect contributions of perivascular stem cells to bone healing

项目摘要 作为自体移植骨的替代物，间充质干细胞（MSC）已被用于加速骨形成。 骨修复然而，当前的MSC源具有显著的缺点，包括：（1）稀缺的可用性，（2） 需要培养，和（3）显著的细胞异质性，骨形成功效降低。我们的解决方案是 使用一种新的、纯化的和未培养的干细胞来源，称为血管周围干细胞（PSC）。 PSC是培养衍生的传统MSC的预期纯化祖先，并通过其特征进行鉴定。 在船只周围。与骨髓干细胞（BMSC）不同，PSC具有显著的优势， 临床转化，包括：（1）通过荧光激活细胞分选（FACS）而不是培养进行分离，（2） 同质性高;（3）临床应用量高。 我们的数据表明，PSC通过直接和旁分泌机制发挥骨愈合作用，包括 结合对宿主骨组织的促成骨和促血管生成作用。令人振奋的最新数据表明， PSC频率、活力、生长和分化因子（GDF）的加工和成骨分化 对骨质疏松性骨丢失的有害影响有抵抗力。相比之下，BMSC显示数量减少， 增殖和成骨分化潜能与骨质疏松症。这种显著的差异使我们 直接和系统地比较PSC与BMSC在当前提案中的骨愈合潜力。 总体而言，以下早期/新研究者申请将解决细胞和信号传导问题， 血管周围干细胞通过直接和间接作用促进骨愈合的机制， 以及它们对干旱条件的独特抵抗力。 目的1：评价PSC在骨愈合中的直接和旁分泌作用。通过系统的系列 我们将研究PSC对骨祖细胞迁移和分化的影响，以及 骨愈合中的血管生成和新血管形成。成骨细胞和内皮细胞报告动物将 用于比较我们先前描述的股骨节段性缺损（FSD）中宿主：供体细胞的贡献 模型研究将采用培养衍生的BMSC应用程序进行。骨差异 治疗组之间的愈合结果将与体外GDF加工在统计学上相关。 目的2：证明宿主与供体骨质疏松症在PSC骨愈合中的重要性。作为 如前所述，PSC对骨质疏松性骨丢失的有害影响表现出显著的抵抗力。 然而，我们的试验数据表明，“宿主骨”的增生状态而不是“供体MSC”可能影响 骨愈合在这里，我们将确定移植PSC或宿主骨中骨质疏松的程度， 组织影响骨愈合。研究将与培养来源的BMSC应用进行头对头， 我们的消防处模型骨愈合的差异将与GDF和骨抑制信号表达相关。

PROJECT SUMMARY As an alternative to autograft bone, mesenchymal stem cells (MSC) have been employed to accelerate bone repair. However, current MSC sources have significant drawbacks, including: (1) scarce availability, (2) need for culture, and (3) significant cell heterogeneity with decreased bone-forming efficacy. Our solution is the use of a novel, purified and uncultured stem cell source, known as Perivascular Stem Cells (PSC). PSC are prospectively purified ancestors of culture-derived, traditional MSC, and are identified by their presence around vessels. Unlike bone marrow stem cells (BMSC), PSC have significant advantages for clinical translation, including: (1) isolation by fluorescence activated cell sorting (FACS) rather than culture, (2) high homogeneity, and (3) high quantity for clinical application. Our data show that PSC exert bone healing effects via both direct and paracrine mechanisms, including a combination of pro-osteogenic and pro-vasculogenic effects on host bone tissue. Exciting recent data suggest that PSC frequency, viability, growth and differentiation factor (GDF) elaboration and osteogenic differentiation are resistant to the detrimental effects of osteoporotic bone loss. In contrast, BMSC show reduced numbers, proliferation, and osteogenic differentiation potential with osteoporosis. This marked difference has led us to directly and systematically compare the bone healing potential of PSC versus BMSC in the current proposal. Overall, the following Early Stage / New Investigator application will address the cellular and signaling mechanisms through which perivascular stem cells promote bone healing through direct and indirect effects, and their unique resistance to osteoporotic conditions. AIM 1: Evaluate the direct and paracrine effects of PSC in bone healing. Through a systematic series of studies, we will examine the effects of PSC on osteoprogenitor cell migration and differentiation, as well as angiogenesis and neovascularization in bone healing. Osteoblast and endothelial cell reporter animals will be utilized to compare host:donor cell contributions in our previously described femoral segmental defect (FSD) model. Studies will be performed head-to-head with culture-derived BMSC application. Differences in bone healing outcomes between treatment groups will be statistically correlated with in vitro GDF elaboration. AIM 2: Demonstrate the importance of host versus donor osteoporosis in PSC bone healing. As mentioned, PSC demonstrate marked resistance to the detrimental effects of osteoporotic bone loss. However, our pilot data suggest that osteoporotic status of the ‘host bone’ but not ‘donor MSC’ may impact bone healing. Here, we will determine the extent to which osteoporosis within transplanted PSC or host bone tissue affects bone healing. Studies will be performed head-to-head with culture-derived BMSC application in our FSD model. Differences in bone healing will be correlated with GDF and bone inhibitory signal expression.