Enhancing Bcl-2 Expression for Bone Regeneration.

描述（由申请人提供）：颅面骨缺损的重建，无论是先天性的还是后天性的，都是一项艰巨的挑战。当前的策略采用骨移植和游离组织移植，但供体部位的发病率以及形态和功能的次优恢复继续推动新方法的发展。再生医学领域对解决这一需求有着重大的希望，采用基于细胞的策略来替代受损或缺陷的组织。然而，尽管取得了巨大的进步，仍然存在一些复杂的问题。干细胞的临床应用通常涉及放置在缺氧和具有显著上调炎症介质的敌对伤口中。在如此恶劣的环境中，细胞存活是不确定的，植入细胞的效用可能会严重受损。因此，越来越多的人认识到，仅仅将干细胞植入伤口不足以确保最大限度的组织再生。虽然研究人员一直专注于促进血管生成或限制炎症，但一种新的方法是通过上调Bcl-2（一种细胞凋亡调节剂）来增强移植细胞本身的促存活途径，从而促进骨再生。重要的是，Bcl-2的表达增加可以减少间充质细胞的凋亡，而不会限制其随后的分化能力。然而，Bcl-2的有限表达是必要的，以避免任何致瘤风险。因此，我们将采用一种非整合的、非病毒的小圆环载体来转染人脂肪来源的基质细胞，并评估其后续的生存能力和骨形成能力。此外，为了最大限度地发挥我们方法的转化潜力，我们将开发一种创新的支架，将聚乙烯亚胺包裹的超顺磁性氧化铁纳米颗粒与我们表达Bcl-2的微环络合在一起。这将允许一种“现成”的方法，即新鲜收获的细胞可以直接播种到支架上，然后放回患者体内进行原位转染。我们将评估这种新策略的能力，以提高人类脂肪来源的基质细胞植入免疫功能低下小鼠的非愈合临界大小的颅骨缺陷的存活率和骨再生能力。总的来说，所提出的实验将确定Bcl-2上调促进骨修复的能力，并促进修复大型骨骼缺陷的新临床应用的发展。

DESCRIPTION (provided by applicant): Reconstruction of craniofacial skeletal defects from both congenital and acquired etiologies can often present a daunting challenge. Contemporary strategies employ bone grafting and free tissue transfer, but donor site morbidity, as well as suboptimal restoration of form and function, continues to drive the development of novel approaches. The field of regenerative medicine holds significant promise to address this need, employing cellular-based strategies to replace damaged or deficient tissues. Despite tremendous advances, however, several complex issues remain. Clinical application of stem cells often involve placement into hostile wounds that are hypoxic and possess dramatically upregulated inflammatory mediators. Within such a harsh environment, cellular survival is uncertain, and the utility of implanted cells can be severely compromised. In light of this, there s growing recognition that simple placement of stem cells into a wound is not sufficient to ensure maximal tissue regeneration. While investigators have focused on promoting angiogenesis or limiting inflammation, a novel approach would be to enhance pro-survival pathways within the transplanted cells themselves through upregulation of Bcl-2, a regulator of apoptosis, for bone regeneration. Importantly, increased expression of Bcl-2 has been shown to reduce apoptosis without limiting subsequent differentiation capacity in mesenchymal cells. However, limited expression of Bcl-2 is necessary to avoid any tumorigenic risk. We will therefore employ a non-integrating, non-viral minicircle vector to transfect human adipose-derived stromal cells and evaluate their subsequent viability and bone forming capacity. Furthermore, to maximize the translational potential of our approach, we will develop an innovative scaffold incorporating polyethylenimine- encapsulated superparamagnetic iron oxide nanoparticles complexed to our Bcl-2 expressing minicircle. This will allow for an "off the shelf" approach where freshly harvested cells can be directly seeded onto scaffolds and placed back into the patient for in situ transfection. We will evaluate the ability for this novel strategy to enhance survival and bone regenerative ability of human adipose-derived stromal cells implanted into non- healing critical-sized calvarial defects in immunocompromised mice. Collectively, the experiments proposed will determine the ability for Bcl-2 upregulation to promote bone repair and facilitate development of new clinical applications to repair large skeletal defects.