Enhancing Bcl-2 Expression for Bone Regeneration.

描述(由申请人提供)：先天和后天原因造成的颅面部骨骼缺陷的重建通常是一个艰巨的挑战。当代的治疗策略采用了骨移植和游离组织移植，但供体部位的发病率以及形状和功能的次优恢复，继续推动着新方法的发展。再生医学领域很有希望满足这一需求，采用基于细胞的策略来替换受损或缺陷的组织。然而，尽管取得了巨大进展，仍存在几个复杂的问题。干细胞的临床应用通常涉及将其植入低氧和具有显著上调的炎症介质的敌意伤口。在如此恶劣的环境中，细胞的存活是不确定的，移植细胞的效用可能会受到严重影响。有鉴于此，S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，但一种新的方法将是通过上调促进骨再生的细胞凋亡调节因子Bcl-2来增强移植细胞本身的促生存途径。重要的是，Bcl2表达的增加被证明可以减少间充质细胞的凋亡，而不会限制随后的分化能力。然而，为了避免任何肿瘤发生的风险，有限的Bcl-2表达是必要的。因此，我们将使用一种非整合、非病毒的微环载体来转染人脂肪来源的基质细胞，并评估其后续的活性和成骨能力。此外，为了最大限度地发挥我们方法的翻译潜力，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的表达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种植到支架上，然后放回患者体内进行原位转基因。我们将评估这一新策略的能力，以提高人类脂肪来源的基质细胞移植到免疫受损小鼠无法愈合的临界大小的颅骨缺损中的存活和骨再生能力。总之，拟议的实验将确定Bcl2上调促进骨修复的能力，并促进修复大型骨缺损的新临床应用的开发。

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.