Engineering Differentiation of Multi-tissue Units

描述（由申请人提供）：用于驱动干细胞朝向肌肉骨骼系统的空间组织的多组织单元（如肌肉-肌腱-骨（MTB））的组织工程方法将需要对由组织工程构建体的各种组分提供的分化线索进行空间控制，包括其：生化元素;支架材料组成和结构;以及生物力学相互作用。目前用于帮助发现、设计和实施这种复杂的多变量构建体的早期阶段的工具集要么不存在，要么在其并入由外源旁分泌信号传导因子（PSF）提供的那些生化元件的空间控制的能力方面受到严重限制。为了满足这一需求，我们提出了一种新的PSF生物图案化技术，该技术将能够创建在支架的多个相邻区域中组织的持久的、空间定义的PSF图案，其中每个区域靶向待诱导的不同表型。这种能力将是独特的，因为它将使暴露于PSF图案化构建体的外源性或内源性干细胞群能够在亚毫米分辨率下被驱动朝向与这些图案同时配准的多种分化命运，以在体外和体内在同一构建体内形成相邻的多表型分组。MTB的图案设计将首先借助于应用于体外研究的系统设计方法来确定，以从非常大量的设计可能性中识别出空间图案化的PSF线索的最小集合，然后将在体内验证所得的最高等级设计以驱动异位皮下小鼠模型中的组织表型形成。作为额外的体内验证，将PSF图案化的构建体植入小鼠跟腱伤口模型中以引发位点特异性宿主应答，并在组织学上评估与所应用的图案配准的组织表型表达。 公共卫生相关性：需要新的组织工程疗法来解决日益增长的修复肌肉骨骼系统的多组织结构的需求，例如患病或受伤的相互连接的骨-肌腱-肌肉单元。这成为一个更大的挑战，因为需要空间控制多个分化命运同时，包括多单元组织界面，在同一个相互沟通的细胞周围环境。存在对新的组织工程化构建体技术和设计方法的未满足的需求，所述技术和设计方法将使得干细胞群能够在体外和体内被驱动朝向每个区域中不同分化命运的相邻区域。我们建议开发和展示一种空间图案化方法，该方法使用有限数量的外源性信号分子，在支架中图案化，将肌肉骨骼系统中的干细胞引导到多个相邻和相互交流的分化命运，作为多组织形成和相互作用的一阶模型。工程化空间图案化将提供关于多组织形成的新见解，其长期目标是使用图案化构造来改善肌肉组织相关治疗的临床结果，这表示估计每年直接和间接成本为5100亿美元，以2004年美元计，或仅美国GDP的3.1%。

DESCRIPTION (provided by applicant): Tissue engineering approaches for driving stem cells toward spatially-organized multi-tissue units of the musculoskeletal system, such as muscle-tendon-bone (MTB), will require spatial control of differentiative cues provided by various components of tissue-engineered constructs, including their: biochemical elements; scaffold material composition and structure; and, biomechanical interactions. Current toolsets to aid in the early stages of discovery, design, and implementation of such complex, multi-variable constructs are either non- existent or severely limited in their capabilities to incorporate spatial control of those biochemical elements provided by exogenous paracrine signaling factors (PSFs). To address this need, we propose a novel PSF biopatterning technology that will enable the creation of persistent, spatially-defined patterns of PSFs organized in multiple neighboring regions of a scaffold, where each region targets a different phenotype to be induced. This capability will be unique because it will enable an exogenous or endogenous stem cell population exposed to a PSF-patterned construct to be driven toward multiple differentiative fates simultaneously in register to these patterns, at sub-millimeter resolution, to form neighboring multi-phenotype groupings within the same construct, both in vitro and in vivo. Pattern designs for an MTB will first be determined with the aid of a systematic design methodology applied to in vitro studies to identify a minimum set of spatially-patterned PSF cues out of a very large number of design possibilities, and then the resulting highest ranking designs will be validated in vivo for driving tissue phenotype formation in an ectopic subcutaneous mouse model. As an additional in vivo validation PSF patterned constructs will be implanted into a mouse Achilles tendon wound model to initiate site-specific host response, and histologically assessed for tissue phenotype expression in register to patterns applied. PUBLIC HEALTH RELEVANCE: New tissue engineering therapies are needed to address the growing demand to repair multi-tissue structures of the musculoskeletal system, such as interconnected bone-tendon-muscle units that are diseased or injured. This becomes an even greater challenge because of the need to spatially control multiple differentiation fates simultaneously, including multi-unit tissue interfaces, within the same intercommunicating pericellular environment. There is an unmet need for new tissue-engineered construct technologies and design methodologies that will enable a stem cell population to be driven toward neighboring regions of different differentiation fates in each region, in vitro and in vivo. We propose to develop and demonstrate a spatial patterning methodology that uses a limited number of exogenous signaling molecules, patterned in scaffolds, to direct stem cells in the musculoskeletal system down multiple neighboring and intercommunicating differentiation fates as a first order model of multi-tissue formation and interaction. Engineered spatial patterning will provide new insights about multi-tissue formation, with the long-term goal to use patterned constructs to improve clinical outcomes of musculoskeletal-related treatments, which represents an estimated annual direct and indirect cost of $510 billion, in terms of 2004 dollars, or 3.1 % of the GDP in the US alone.