Engineering Differentiation of Multi-tissue Units

描述(申请人提供)：将干细胞驱动为肌肉骨骼系统的空间组织的多组织单元的组织工程方法，如肌肉-肌腱-骨(MTB)，将需要对组织工程构造的不同组件提供的分化提示进行空间控制，包括：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结构的早期阶段的工具集要么不存在，要么严重限制了它们整合由外源旁分泌信号因子(PSF)提供的那些生化元件的空间控制的能力。为了满足这一需求，我们提出了一种新颖的PSF生物调制技术，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模式，其中每个区域针对要诱导的不同表型。这种能力将是独特的，因为它将使暴露于PSF图案构建物的外源或内源干细胞群体在体外和体内以亚毫米分辨率注册这些模式，以在相同的构建物内形成相邻的多表型组，从而同时驱动暴露于PSF图案的构建物的多个分化命运。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PSF线索的最小集合，然后将在体内验证所得到的最高排名的设计以驱动异位皮下小鼠模型中的组织表型形成。作为另一项体内验证，PSF图案化构建物将被植入小鼠跟腱创伤模型，以启动特定部位的宿主反应，并根据应用的图案库对组织表型表达进行组织学评估。 公共卫生相关性：需要新的组织工程疗法来满足日益增长的修复肌肉骨骼系统多组织结构的需求，例如疾病或受伤的相互连接的骨-肌腱-肌肉单位。这成为一个更大的挑战，因为需要在同一个相互通信的细胞周围环境中同时在空间上控制多个分化命运，包括多个单位组织界面。对于新的组织工程化构建技术和设计方法的需求尚未得到满足，这些技术和设计方法将使干细胞群体能够在每个区域的不同分化命运的邻近区域，在体外和体内。我们建议开发和演示一种空间构图方法，该方法使用有限数量的外源信号分子在支架中构图，以引导肌肉骨骼系统中的干细胞沿着多个相邻的和相互通信的分化命运向下移动，作为多组织形成和相互作用的一级模型。工程化空间模式将提供有关多组织形成的新见解，长期目标是使用图案化结构来改善肌肉骨骼相关治疗的临床结果，按2004年美元计算，估计每年直接和间接成本为5100亿美元，仅在美国就占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.