Enhancement Of Bone Regeneration From Human Adipose-Derived Stromal Cells

描述（由申请人提供）：2006年，来自美国健康成本与利用项目的数据显示，超过100万例骨科相关手术的成本超过70亿美元，这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（ASCs）分化成骨的能力表明，它们可能被用于满足遗传疾病、退行性疾病和创伤或手术后骨骼组织缺损患者日益增长的需求。虽然类似的技术可以用于骨髓细胞和胚胎干细胞，但脂肪细胞更容易大量获得，并且缺乏伦理问题。最终的转化目标是能够在一次手术室内收集脂肪组织，获得足够数量的人类ASCs，分离出具有更大成骨潜力的细胞，并将这些细胞植入骨骼缺陷。下面的建议将促进一种新的手段的发展，以促进骨骼缺损患者快速和强健的骨形成。中心假设是，人类脂肪来源的基质细胞代表了一个容易获得的细胞群，用于设计骨骼再生的细胞疗法。在第一个具体目标中，将确定体外具有增强成骨能力的人类ASC亚群。初步研究表明，细胞表面标记CD105在小鼠中具有增强的成骨潜能，该标记将用于鉴定具有增强成骨能力的人类细胞。人体脂肪组织将使用标准吸脂技术进行采集。基于细胞表面标记物（cd105高和cd1050低），利用荧光活化细胞分选（FACS）分离具有增强成骨潜能的人ASCs。这两个亚群的成骨潜能将通过QT-PCR和茜素红染色进行分析。在第二个特定目标中，通过使用PLGA支架将具有增强成骨潜能的人类ASCs亚群植入骨骼缺陷，将在体内加速骨骼愈合。骨愈合将评估和量化使用显微计算机断层扫描（microCT），详细组织学，组织形态计量学和荧光原位杂交（FISH）。从这些目标中获得的数据将展示一种修复和再生受创伤、疾病、手术或畸形影响的骨骼的创新策略，这与NIH减轻残疾负担的使命是一致的。

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.