Nano-Ceramic for Metabolic Stem Cell Engineering

描述(申请人提供)：该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，使用纳米晶陶瓷支架，用于增殖脂肪来源的前体细胞并将其分化为成骨细胞。纳米羟基磷灰石(Nano-HAP)支架的使用为骨组织工程提供了几个优点：1)新出现的证据表明，纳米相基质增强了锚定依赖细胞的黏附和黏附依赖功能；2)纳米羟基磷灰石的陶瓷特性为支架提供了化学稳定性，因此具有长期形状完整性的优势；3)支架机械性能坚固，能在植入过程中和体内支持和保护骨组织构建，从而实现培养和植入步骤的一体化。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。与骨髓细胞不同，脂肪细胞很容易获得，对患者的不适感最小。这一阶段的STTR项目利用纳米HAP独特的材料特性和代谢工程中强大的细胞特性分析和设计工具，确定了脂肪MSCs体外扩增和分化为成骨细胞的最佳支架参数和培养条件。这项工作假设纳米羟基磷灰石的表面形貌促进了细胞膜和基质蛋白之间的相互作用，从而提高了贴壁细胞的稳定性，从而改善了黏附依赖细胞的功能。其具体目标是：1)脂肪来源的成体干细胞在理想化的2D纳米表面上扩增；2)诱导分化的成骨功能标志物；3)通过表面性质的改变来定向调节成骨细胞的功能。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类脂肪前体细胞在纳米羟基磷灰石和其他常用支架材料上的黏附和增殖。这些实验还将评估不同的纳米羟基磷灰石颗粒和孔径大小在有或没有基质蛋白预处理的情况下的效果。其次，在剂量效应实验之后，将进行析因设计实验，以系统地探索已知的分化因素空间。一个新开发的生物信息学工具将被用来在功能上将分化因子剂量和组合与分化的程度和速度联系起来。重点将放在通过控制向脂肪细胞的分化来选择性地向成骨细胞分化。最后，将进行代谢流量分析，以调查中间代谢可能被引导以支持分化的成骨细胞功能的假设，包括基质蛋白沉积和骨形成(矿化)。利用上述生物信息学工具，纳米羟基磷灰石的表面性质、代谢通量分布和成骨细胞活性将在功能上相关。该项目有望通过进一步的代谢工程来确定特定的支架属性、介质因子或酶靶标，以增强成骨细胞的功能。

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.