Muscle-based tissue engineering to improve bone healing

颅面部骨骼骨缺损的不完全愈合是常见的。成骨蛋白，包括骨形态发生蛋白2和4(BMP2，BMP4)，可以促进骨缺损的愈合，但这些蛋白的半衰期短，血流清除快，限制了它们的应用。我们最初的R01和第一个竞争性更新项目的主要目标是开发基于肌肉来源干细胞(MDSCs)的组织工程方法，以有效地输送成骨蛋白并改善颅面骨愈合。简而言之，在这个资助期间，我们证明了通过基因工程表达BMP2和BMP4的MDSCs向成骨方向分化，并可以促进颅骨和长骨缺损处的骨愈合。 我们还发现，同时表达血管内皮生长因子(VEGF)可以改善BMP2和13MP4表达的MDSCs植入后的骨愈合。此外，我们还证实了供体性别对小鼠骨髓间充质干细胞的体外成骨能力和体内骨再生能力的影响，并证实了基因工程和骨形态发生蛋白信号通路的操作可以提高骨髓间充质干细胞的成骨能力。最后，我们分离出了与小鼠骨髓间充质干细胞相当的人类干细胞，并在体外测定了它们的成骨能力。我们要感谢NIDCR在前一个供资期间提供的支持。我们达到并超过了最初的R01申请以及第一次竞争更新中的所有关键目标，我们的结果构成了33篇论文和117篇摘要的基础。这份DE013420第二份竞争性续订申请概述了旨在扩展这些初步发现并导致以下结果的实验。MDSCs未来可能在临床上应用于改善骨愈合。我们将把第二次竞争性更新的重点放在人类与小鼠MDSC的等价物上，以及 优化其在骨再生中的应用。由于骨髓间充质干细胞植入包括骨骼在内的受损肌肉骨骼组织后，修复过程通常由宿主细胞的化学吸引所介导，我们计划确定供体细胞在骨愈合过程中对宿主细胞(尤其是血管壁祖细胞)的化学吸引的影响(目标1)。我们计划检测年龄和性别捐献者对该患者来源的hMDCs数量和成骨能力的影响。然后，我们将调查如何 使用基于hMDC的组织工程优化骨形成和愈合，包括通过抑制ERK1/2、pj8 MAPK和PI3K通路以及植入前hMDCs的机械刺激来调节BMP信号(目标2)。拟议的实验将提供关于hMDCs的基本生物学及其用于骨愈合的重要信息，并进一步开发骨缺陷的临床治疗方法。

Incomplete healing of bone defects ih the craniofacia.l skeleton is common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, BMP4), promote healing in bone defects, but the proteins' short half-lives and rapid clearance by the bloodstream limit their utility. The main goal of our initial R01 and the first competitive renewal project was the development of tissue engineering approach'es, based on muscle-derived stem cells (MDSCs), to efficiently deliver osteogenic proteins and improve craniofacial bone healing. In brief, during this funding period, we demonstrated that MDSCs genetically engineered to express BMP2 and BMp4 differentiate toward an osteogenic lineage and can improve bone healing in calvarial and long bone defects. We also found that concomitant expression Of vasCular endothelial growth factor (VEGF) improves the bone healing observed after implantation Of BMP2 and 13MP4 expressing MDSCs. Additionally, we have demonstrated that donor sex influences the in vitro osteogenic potential and in vivo bone regeneration potential of murine MDSCs and also identified wa.ys, such as genetic engineering and manipulation of the BMP signaling pathways, to improve the osteogenic potential of MDSCs. Finally, we have isolated the human equivalents of the murine MDSCs and determine their osteogenic potential in vitro. We would like to thank NIDCR for their support during the prior funding period. We met and exceeded all the key objectives in the original R01 application as well as the first competitive renewal, and our results formed the basis for 33 + papers and 117+ abstracts. This DE013420 second cornpetitive renewal application outlines experiments designed to extend these initial findings and lead to. possible future clinical applications of MDSCs to improve bone healing. We will focus this second competitive renewal on human equivalents to murine MDSCs and optimization of their use for bone regeneration. Since after implantation of MDSCs into injured musculoskeletal tissues, including bone, the repair process is often mediated by chemoattraction of host cells, we plan to determine the influence of host cells(especially blood vessel wall progenitors) chemoattraCted by donor human cells during the bone healing process (Aim 1). We plan to examine the effect of age and sexotdonor patient on the number and osteogenic potential of hMDCs derived from that patient. We then will investigate ways to optimize bone formation and healing by using hMDC-based tissue engineering, including the modulation of BMP signaling through inhibition of ERK1/2, pj8 MAPK and PI3K pathwa.ys and mechanical stim'ulation of hMDCs prior to implantation (Aim 2). The proposed experiments will provide important information regarding the basic biology of hMDCs and their use for bone healing and further the development of clinical treatments for osseous deficiencies.