Patterning instructive biomolecular cues into collagen scaffolds for tendon inser

描述（由申请人提供）：空间有序的多组织结构，如肌腱-骨连接（TBJ），对组织工程领域提出了独特的、未满足的挑战。TBJ包含连接肌腱和骨间室的分级界面区，这对其机械能力至关重要，但也是常见的损伤部位。临床干预后经常发生再撕裂，因为目前的手术策略放弃了界面再生（生物固定），将肌腱机械固定到骨上。分级的建筑和生化线索的形态发生，重塑和功能的多组织结构，如TBJ的关键建议，概括其空间有序的设计可能是再生医学应用的重要功能。为了产生能够引导附着点再生的单一复合生物材料，我们提出探索生物启发的结构和生长因子线索如何使用独特的胶原-GAG（CG）支架平台诱导空间选择性、多谱系间充质干细胞（MSC）分化。我们假设生物材料结构（各向异性，矿物质含量）和生化（栓系生长因子）信号的重叠模式是肌腱和骨之间连续解剖插入的功能再生所需的。因此，本提案的目的是研究空间分级结构和生化信号可以协同诱导多谱系MSC分化的机制。我们将利用包含不同的腱（非矿化，各向异性）和骨（矿化）隔室的原型TBJ支架来研究MSC整合物理（支架各向异性，矿物质含量）和生物化学（GDF-5，BMP-2）线索以产生对一系列独特细胞微环境的协调反应的机制。目的1将确定支架结构和生物分子线索对附着点特异性分化谱的自催化诱导的综合影响。在这里，我们将研究不同的支架隔室是否诱导机械转导和TGF-β超家族信号通路的协同激活，以偏向多谱系hMSC分化谱。目的2将研究区域不同的新TBJ组织形成的空间组织的微观结构和生物分子线索的贡献。我们将使用体外生物反应器和体内小鼠骨腱再生模型评估新附着点组织结构的形成。这种先进的还原方法使我们能够复制天然TBJ的潜在调控元件，同时保留操纵生物材料结构和生物分子的能力。 以确定的增量提示。这些信息对于设计基于生物材料的变阻器以在紧密空间配准中控制多个hMSC命运决定将是至关重要的。最终，这种方法可以识别能够在植入前立即用自体hMSC接种的支架变体，以再生稳定的肌腱-骨附着点。

DESCRIPTION (provided by applicant): Spatially-ordered multi-tissue structures such as the tendon-bone junction (TBJ) present unique, unmet challenges for the field of tissue engineering. The TBJ contains a graded interfacial zone linking tendinous and osseous compartments that is critical for its mechanical competence but is also a common injury site. Re-tears are frequent after clinical intervention because current surgical strategies forsake interface regeneration (biological fixation) for mechanical fixation of tendon to bone. The graded architectural and biochemical cues critical for the morphogenesis, remodeling, and function of multi-tissue structures such as the TBJ suggest that recapitulating their spatially-ordered design may be of significant functional importance for regenerative medicine applications. With the goal of generating a single composite biomaterial capable of guiding enthesis regeneration, we propose to explore how bioinspired structural and growth factors cues can induce spatially- selective, multi-lineage mesenchymal stem cell (MSC) differentiation using a unique collagen-GAG (CG) scaffold platform. We hypothesize that overlapping patterns of biomaterial structural (anisotropy, mineral content) and biochemical (tethered growth factors) signals are required for functional regeneration of the continuous anatomic insertion between tendon and bone. The objective of this proposal is therefore to investigate mechanisms by which spatially-graded structural and biochemical signals can synergistically induce multi-lineage MSC differentiation. We will leverage a prototype TBJ scaffold containing distinct tendinous (non- mineralized, anisotropic) and osseous (mineralized) compartments to investigate mechanisms by which MSCs integrate physical (scaffold anisotropy, mineral content) and biochemical (GDF-5, BMP-2) cues to generate a coordinated response to a series of unique cellular microenvironments. Aim 1 will define the combined influence of scaffold structural and biomolecular cues on autocatalytic induction of enthesis-specific differentiation profiles. Here, we will examine whether distinct scaffold compartments induce synergistic activation of mechanotransduction and TGF-¿ superfamily signaling pathways in order to bias multi-lineage hMSC differentiation profiles. Aim 2 will examine the contribution of spatially-organized microstructural and biomolecular cues on regionally-distinct neo-TBJ tissue formation. We will assess formation of neo-enthesis tissue structures using both an in vitro bioreactor and an in vivo murine osteotendinous regeneration model. This advanced reductionist approach allows us to replicate potential regulatory elements of the native TBJ while retaining the ability to manipulate biomaterial structural and biomolecular cues in defined increments. Such information will be critical for designing biomaterial-based rheostats to control multiple hMSC fate decisions in close spatial register. Ultimately this approach may identify scaffold variants able to be seeded with autologous hMSCs immediately prior to implantation in order to regenerate a stable tendon-bone enthesis.