Cranial Bone Repair with Adipose-Derived Stem Cells

描述(申请人提供)：这项建议的长期目标是开发一种有效的治疗方法，利用来自脂肪组织的成人干细胞修复头面部骨骼中的骨缺陷。对于发育异常、创伤和肿瘤切除后的重建手术，需要材料来替代或诱导骨再生。目前应用的方法，如使用自体移植物和银行骨，在疗效上是有限的，并带有供区并发症和感染的风险。替代骨的组织工程学在提供明显优势的同时，缺乏现有方法的局限性。脂肪来源的成体干细胞(ADASC)易于分离，可在体外诱导分化为包括骨祖细胞在内的多种神经和中胚层细胞系。这项研究的中心假设是ADASCs可以被维持为复制多潜能干细胞，在体外可以被诱导分化为成骨细胞，当适当地输送时，将修复体内的骨缺陷。为了验证这一假说，提出了两个特定的目标：1)鉴定和优化ADASCs的体外分离、生长和分化；2)测试人ADASCs介导修复无菌大鼠颅骨临界大小骨缺损的能力。在目标1中，将确定ADASC总分离株中骨祖细胞群体的克隆性质以及骨特异基因表达的时间。利用成骨分化的早期和晚期标记物，分化条件将被优化，并与原代人类成骨细胞和来自人类胚胎干细胞系的成骨前体细胞进行比较。在AIM 2中，ADASCs将以胶原海绵的形式输送。然后，在愈合过程中，通过计算机断层扫描(3DCT)的三维重建来测量新生骨的形成，以确定骨修复的最佳细胞和传代数量、分化阶段和种植密度。这些研究应该表明ADASC对骨组织工程做出贡献的潜力，并是测试正在开发的许多组织支架系统和使用干细胞作为基因载体的方法的先决条件。这些特性研究也可能为将该干细胞系统用作成骨分化研究的模型提供基础。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop an effective therapy to repair bony defects in the craniofacial skeleton employing adult stem cells derived from adipose tissue. Reconstructive surgery for developmental anomalies, trauma and resection following tumor removal requires materials to replace or induce the regeneration of bone. Currently applied methods, such as use of autologous grafts and banked bone, are limited in efficacy and carry the risk of donor site morbidity and infection. Tissue engineering of replacement bone lacks the limitations of current approaches while offering distinct advantages. Adipose-derived adult stem cells (ADASC) are easily isolated and can be induced to differentiate ex vivo into a variety of neural and mesodermal cell lineages including osteoprogenitors. The central hypothesis of this study is that ADASCs can be maintained as replicating multipotent stem cells, which can be induced to differentiate into osteoblasts in vitro and, when delivered appropriately, will repair bony defects in vivo. Two Specific Aims are proposed to test the hypothesis: 1) to characterize and optimize the isolation, growth and differentiation of ADASCs in vitro; and 2) to test the capacity of human ADASCs to mediate repair of critical-sized bone defects in the skulls of athymic rats. In Aim 1 the clonal nature of the osteoprogenitor population within the total ADASC isolate and the timing of expression of bone specific genes will be determined. Using early and late markers of osteogenic differentiation, conditions for differentiation will be optimized and compared with primary human osteoblasts and osteoprogenitors derived from a human embryonic stem cell line. In Aim 2, ADASCs will be delivered in collagen sponges. Then de novo bone formation will be measured by three-dimensional reconstruction of computed tomography (3DCT) at intervals over the healing process to determine the optimal cell and passage number, stage of differentiation, and seeding density for bone repair. These studies should indicate the potential of the ADASC to contribute to bony tissue engineering and are a prerequisite to testing of the many tissue scaffold systems being developed and methods for using stem cells as gene delivery vehicles. The characterization studies may also provide the basis to use this stem cell system as a model for study of osteogenic differentiation.