Engineering Differentiation of Multi-tissue Units

描述（由申请人提供）：组织工程方法用于驱动干细胞向肌肉骨骼系统的空间组织多组织单位，如肌肉-肌腱-骨（MTB），将需要对组织工程构建的各种组成部分提供的分化线索进行空间控制，包括：生化元素；支架材料组成及结构；生物力学的相互作用。目前用于早期发现、设计和实施这些复杂、多变量结构的工具集要么不存在，要么在整合外源性旁分泌信号因子（psf）提供的生化元素的空间控制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生物图谱技术，该技术将能够在支架的多个相邻区域组织PSF的持久的、空间定义的模式，其中每个区域针对不同的表型进行诱导。这种能力将是独一无二的，因为它将使外源性或内源性干细胞群体暴露于psf模式构建中，在亚毫米分辨率下，同时被驱动向多个分化命运，在同一构建中形成相邻的多表型组，在体外和体内都是如此。MTB的模式设计将首先通过应用于体外研究的系统设计方法来确定，以从大量的设计可能性中确定最小的空间模式PSF提示集，然后得到的最高排名的设计将在体内验证，以驱动异位皮下小鼠模型的组织表型形成。作为一项额外的体内验证，PSF模式构建体将被植入小鼠跟腱损伤模型中，以启动部位特异性宿主反应，并对应用模式的组织表型表达进行组织学评估。

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.