Nano-Ceramic for Metabolic Stem Cell Engineering

描述（由申请人提供）：本项目的总体目标是开发一种临床上和商业上可行的组织工程化骨结构，其使用纳米晶陶瓷支架将脂肪源性祖细胞增殖和分化为成骨谱系细胞。纳米结构羟基磷灰石的用途纳米羟基磷灰石（nano-HAP）支架为骨组织工程提供了几个优点：1）新的证据表明纳米相基质增强了锚定依赖性细胞的粘附和粘附依赖性功能; 2）纳米羟基磷灰石的陶瓷性质为支架提供了化学稳定性，因此赋予了长期形状完整性的优点; 3）支架在机械上是坚固的，并且可以在植入过程中和在体内支撑和保护骨组织构建体，从而允许培养和植入步骤的整合。越来越多的证据表明，人类脂肪组织中含有能够分化为成骨细胞的间充质干细胞（MSCs）。与骨髓细胞不同，脂肪细胞很容易获得，并导致患者不适最小。该I期STTR项目利用nano-HAP的独特材料特性，对抗来自代谢工程的强大细胞特性分析和设计工具，以确定脂肪MSC体外扩增和分化成成骨细胞的最佳支架参数和培养基条件。这项工作假设纳米HAP的表面形貌促进细胞膜和基质蛋白之间的相互作用，导致粘附细胞的更大稳定性，从而改善粘附依赖性细胞功能。具体目标是：1）脂肪来源的成体干细胞在理想化的2D纳米表面上的扩增; 2）诱导分化的成骨功能标志物;和3）通过表面性质修饰定向调节成骨细胞功能。这些具体目标将通过以下研究设计和方法来实现。首先，平行细胞培养实验将比较人脂肪祖细胞在纳米HAP和其他常用支架材料上的粘附和增殖。这些实验还将评估在有或没有基质蛋白预处理的情况下改变纳米HAP颗粒和孔径的影响。其次，剂量反应实验之后将进行析因设计实验，以系统地探索已知的分化因子空间。一个新开发的生物信息学工具将用于功能相关的分化因子剂量和组合的分化程度和速度。重点将放在通过控制分化成脂肪细胞选择性分化成骨细胞。最后，将进行代谢通量分析，以研究中间代谢可能直接支持分化的成骨细胞功能，包括基质蛋白沉积和骨形成（矿化）的假设。纳米HAP表面特性，代谢通量分布，和成骨细胞活性将使用上述生物信息学工具功能相关。该项目预计将确定特定的支架特性，介质因子或酶靶点，通过进一步的代谢工程增强成骨细胞功能。

DESCRIPTION (provided by applicant): The overall objective of this project is to develop a clinically and commercially viable tissue engineered bone construct using nanocrystalline ceramic scaffolds for proliferating and differentiating adipose-derived progenitor cells into cells of the osteogenic lineage. The use of a nanostructured hydroxyapatite (nano-HAP) scaffold offers several advantages for bone tissue engineering: 1) emerging evidence indicates that nanophase substrates enhance the adhesion and adhesion-dependent functions of anchorage-dependent cells; 2) the ceramic nature of nano-HAP affords chemical stability to the scaffold and therefore confers the advantage of long-term shape integrity; 3) the scaffolds are mechanically robust and can support and protect the bone tissue construct during the implantation procedure and within the body, thus allowing integration of culture and implant steps. Increasing evidence shows that human adipose tissue contains mesenchymal stem cells (MSCs) capable of differentiating into osteoblasts. Unlike bone marrow cells, adipose cells are easy to obtain and result in minimal patient discomfort. This Phase I STTR project leverages the unique material properties of nano-HAP against powerful cell property analysis and design tools drawn from metabolic engineering to identify optimal scaffold parameters and medium conditions for in vitro expansion and differentiation of adipose MSCs into osteoblasts. This work hypothesizes that the surface topography of nano-HAP promotes interactions between cell membrane and matrix proteins that result in greater stability of adherent cells, and thus improve adhesion dependent cell functions. The specific aims are: 1) Expansion of adipose-derived adult stem cells on idealized 2D nano-surface; 2) Induction of differentiated osteogenic function markers; and 3) Directed modulation of osteoblast function by surface property modification. These specific aims will be achieved using the following research design and methods. First, parallel cell culture experiments will compare adhesion and proliferation of human adipose progenitor cells on nano-HAP and other commonly used scaffolding material. These experiments will also evaluate the effects of varying nano-HAP grain and pore sizes with or without matrix protein pretreatment. Second, dose response experiments will be followed by a factorial design experiment to systematically explore the known differentiation factor space. A newly developed bioinformatics tool will be used to functionally relate differentiation factor doses and combinations with extent and speed of differentiation. Emphasis will be placed on selective differentiation into osteoblasts by controlling for differentiation into adipocytes. Finally, metabolic flux analysis will be performed to investigate the hypothesis that intermediary metabolism may be directed to support differentiated osteoblast function, including matrix protein deposition and bone formation (mineralization). Nano-HAP surface properties, metabolic flux distribution, and osteoblast activity will be functionally related using the aforementioned bioinformatics tool. This project is expected to identify specific scaffold properties, medium factors, or enzyme targets for enhancing osteoblast function through further metabolic engineering.