Enhancement Of Bone Regeneration From Human Adipose-Derived Stromal Cells

描述（由申请人提供）：2006年，来自美国健康成本和利用项目的数据显示，超过100万例骨科相关手术，成本超过70亿美元，使得使用组织工程进行骨骼修复成为一个有吸引力的目标。脂肪源性基质细胞（ASC）分化成骨的能力表明它们可用于满足患有遗传性疾病、退行性疾病和骨骼的创伤或手术后组织缺陷的患者的不断增加的需求。虽然类似的技术可以用骨髓细胞和胚胎干细胞进行，但脂肪细胞更容易大量获得，而且缺乏伦理问题。最终的转化目标是在一次手术室访问中收获脂肪组织，获得足够数量的人ASC，分离具有更大成骨潜力的细胞并将这些细胞植入骨骼缺损中的能力。下面的建议将促进开发一种新的方法，以促进骨骼缺损患者快速和稳健的骨形成。核心假设是人脂肪来源的基质细胞代表了一个容易获得的细胞群体，用于设计基于细胞的骨骼再生疗法。在第一个具体目标中，将鉴定具有体外成骨能力增强的人ASC亚群。初步研究表明，细胞表面标志物CD 105在小鼠中具有增加的成骨潜力，并且该相同的标志物将用于鉴定具有增加的成骨能力的人类细胞。将使用标准吸脂技术采集人脂肪组织。将使用荧光激活细胞分选术（FACS）基于细胞表面标志物（CD 105高和CD 105低）分离具有增强成骨潜能的人ASC。将使用QT-PCR和茜素红染色分析这两个亚群的成骨潜力。在第二个具体目标中，通过使用PLGA支架将来自具体目标#1的具有增强的成骨潜力的人ASC亚群植入骨骼缺损处，将在体内加速骨骼愈合。将使用显微计算机断层扫描（microCT）、详细组织学、组织形态学和荧光原位杂交（FISH）评估和量化骨愈合。从这些目标获得的数据将证明一种创新的策略，以修复和再生骨已受到创伤，疾病，手术或畸形，这是符合美国国立卫生研究院的使命，以减少残疾的负担。

DESCRIPTION (provided by applicant): In 2006, data from the U.S. Health Cost & Utilization Project revealed over one million orthopedic-related procedures, at a cost in excess of 7 billion dollars making skeletal repair using tissue engineering an attractive target. The ability of adipose-derived stromal cells (ASCs) to differentiate into bone suggests that they may be used to fulfill the mounting needs of patients with genetic disorders, degenerative diseases, and traumatic or post-surgical tissue deficits of the skeleton. Though similar techniques can be performed with bone marrow cells and embryonic stem cells, adipose cells are easier to obtain in large quantities and lack the ethical concerns. The ultimate translational goal would be the ability within one visit to the operating room to harvest adipose tissue, obtain sufficient numbers of human ASCs, isolate those cells with a greater osteogenic potential and implant these cells into the skeletal defect. The proposal below will facilitate the development of a novel means to promote rapid and robust bone formation in patients with skeletal defects. The central hypothesis is that human adipose derived stromal cells represent a readily available population of cells with which to design cell-based therapies for skeletal regeneration. In the first specific aim, a human ASC subpopulation with enhanced osteogenic capacity in vitro will be identified. Preliminary studies have shown the cell surface marker CD105 to have an increased osteogenic potential in mice and this same marker will be used to identify humans cells with increased osteogenic capabilities. Human adipose tissue will be harvested using standard liposuction techniques. Human ASCs with enhanced osteogenic potential will be isolated based on cell surface markers (CD105high and CD105low) using fluorescence activated cell sorting (FACS). The osteogenic potential of these two subpopulations will be analyzed using QT-PCR and Alizarin red staining. In the second specific aim, skeletal healing will be accelerated in vivo by implanting the subpopulation of human ASCs with enhanced osteogenic potential from Specific Aim #1 onto a skeletal defect using a PLGA scaffold. Osseous healing will be assessed and quantified using microcomputed tomography (microCT), detailed histology, histomorphometry and fluorescent in situ hybridization(FISH). Data obtained from these aims will demonstrate an innovative strategy to repair and regenerate bone that has been affected by trauma, disease, surgery or malformations which is consistent with the NIH mission to reduce the burdens of disability.