Muscle-based tissue engineering to improve bone healing

颅面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（BMP 2，BMP 4），促进骨缺损的愈合，但蛋白质的短半衰期和血液的快速清除限制了它们的效用。我们最初的R 01和第一个竞争性更新项目的主要目标是开发基于肌源性干细胞（MDSC）的组织工程方法，以有效地递送成骨蛋白并改善颅面骨愈合。简而言之，在此资助期间，我们证明了经基因工程改造以表达BMP 2和BMP 4的MDSC向成骨谱系分化，并可以改善颅骨和长骨缺损的骨愈合。 我们还发现血管内皮生长因子（VEGF）的同时表达可以改善表达BMP 2和13 MP4的MDSC植入后观察到的骨愈合。此外，我们已经证明供体性别影响小鼠MDSC的体外成骨潜力和体内骨再生潜力，并且还确定了诸如基因工程和BMP信号通路的操纵等方法来提高MDSC的成骨潜力。最后，我们已经分离出了人类的小鼠MDSC的等价物，并确定其在体外的成骨潜力。我们要感谢NIDCR在上一个资助期内的支持。我们达到并超过了原始R 01申请以及首次竞争性更新中的所有关键目标，我们的结果为33 +篇论文和117+篇摘要奠定了基础。该DE 013420第二次竞争性更新申请概述了旨在扩展这些初步发现并导致的实验。MDSC未来可能的临床应用，以改善骨愈合。我们将把第二次竞争性更新集中在鼠MDSC的人类等同物上， 优化其用于骨再生的用途。由于在将MDSC植入受伤的肌肉骨骼组织（包括骨）后，修复过程通常由宿主细胞的化学吸引介导，因此我们计划确定在骨愈合过程中供体人细胞化学吸引的宿主细胞（特别是血管壁祖细胞）的影响（目的1）。我们计划研究年龄和性别对患者来源的hMDCs数量和成骨潜能的影响。然后我们将研究如何 通过使用基于hMDC的组织工程优化骨形成和愈合，包括通过抑制ERK 1/2、p18 MAPK和PI 3 K通路调节BMP信号传导以及在植入前机械刺激hMDC（目的2）。拟议的实验将提供有关hMDCs的基础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.