Enhancing Bcl-2 Expression for Bone Regeneration.

描述（由申请人提供）：先天性和后天性病因引起的颅面骨骼缺损的重建通常是一项艰巨的挑战。当代策略采用骨移植和游离组织转移，但供体部位发病率以及形式和功能的次优恢复继续推动新方法的发展。再生医学领域在解决这一需求方面有着重要的前景，采用基于细胞的策略来替换受损或有缺陷的组织。然而，尽管取得了巨大进展，仍存在一些复杂的问题。干细胞的临床应用通常涉及放置到缺氧并具有显著上调的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。有鉴于此，人们越来越认识到，简单地将干细胞植入伤口并不足以确保最大的组织再生。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（一种凋亡调节剂）来增强移植细胞自身内的促生存途径，以促进骨再生。重要的是，Bcl-2的表达增加已被证明可以减少细胞凋亡，而不限制间充质细胞随后的分化能力。然而，限制Bcl-2的表达是避免任何致瘤风险所必需的。因此，我们将采用非整合的，非病毒的小环载体来转染人脂肪来源的基质细胞，并评估其随后的活力和骨形成能力。此外，为了最大化我们的方法的翻译潜力，我们将开发一种创新的支架，其包含与我们的Bcl-2表达小环复合的聚乙烯亚胺封装的超顺磁性氧化铁纳米颗粒。这将允许“现成”的方法，其中新鲜收获的细胞可以直接接种到支架上并放回患者体内进行原位转染。我们将评估这种新策略增强植入免疫功能低下小鼠非愈合临界尺寸颅骨缺损中的人脂肪源性基质细胞的存活和骨再生能力的能力。总的来说，提出的实验将确定Bcl-2上调促进骨修复的能力，并促进新的临床应用的开发，以修复大的骨骼缺损。

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.