PTHrP/IHH Microenvironment Control for Growth Plate Tissue Engineering

描述(申请人提供)：这项职业发展基金的目的是让我成为一名独立的生物医学科学家，通过在工程微环境模型中研究骨骼发育和疾病来开发组织再生疗法。这项研究的最终目标是组织工程生长板(GP)，以治疗骨骼发育不良和复杂的骨损伤。GP是四肢骨骼末端的软骨结构，它驱动着纵向生长。为了恢复GP和面部和四肢骨损伤患者的肢体长度和几何形状，患者通常需要进行牵张成骨，这是一种使用外部固定器的繁重和漫长(3-6个月)的手术，穿透皮肤，导致巨大疼痛，并有感染和惊吓的风险。使用骨髓干细胞(MSC)来源的组织工程化GP进行骨再生和延长可能被证明比自体移植和去细胞疗法更具侵入性和更有效，因为它具有抗缺氧和招募血管的能力。生长因子甲状旁腺激素相关肽(PTHrP)和印度刺猬(IHH)是通过负反馈梯度环影响GP发生的主要因子。目前尚不清楚PTHrP和IHH梯度是否足以产生GP带状结构。我们假设，PTHrP和IHH激动剂在接种了MSC来源的软骨细胞的水凝胶结构上的反向梯度将诱导细胞分化为GP空间结构。我们将使用微流控生物反应器以及细胞和水凝胶的光修饰来创建一种新的模型。目标1是设计和生成水凝胶中的模型形态梯度分布。我们将在生物反应器内的聚乙二醇二丙烯酸酯(PEGDA)凝胶上产生带有荧光标记的“理想化”形态生物质的单轴梯度。目的2研究软骨细胞的生长糖蛋白样带状分化 在体外生成的构建物与组装的构建物中。我们将在添加软骨细胞的培养液中培养制备MSC来源的软骨细胞。为了创建生成的构建物，我们将分离这些软骨细胞并将其嵌入PEGDA凝胶中，并对凝胶进行PTHrP和IHH激动剂逆梯度和个别梯度的处理。为了创建组装的结构，我们将使用PTHrP和IHH激动剂添加的培养物将软骨细胞预分化为储备和肥大的群体，然后在未处理的细胞的两端进行光培养，形成三层凝胶。目的3是在体内评价工程化GP的发展及其与血管系统的整合。我们将把这些结构植入免疫缺陷小鼠的背部皮下口袋，并使用组织学和显微CT分析来评估结构的生长和GP样结构的维持。

DESCRIPTION (provided by applicant): The purpose of this career development grant is to establish myself as an independent biomedical scientist who develops tissue regeneration therapies through study of skeletal development and disease in engineered microenvironment models. The ultimate goal of the research is to tissue engineer a growth plate (GP) to treat skeletal dysplasia and complex bone injuries. The GP is the cartilaginous structure at the ends of limb bones that drives lengthwise growth. To restore limb length and geometry in patients with GP and compromised facial and extremity bone injuries, patients are often subject to distraction osteogenesis, a burdensome and prolonged (3-6 months) procedure using external fixators that pierce the skin, cause great pain, and risk infection and scaring. Bone regeneration and lengthening using a bone marrow stem cell (MSC) derived tissue engineered GP may prove less invasive and more effective than autografts and acellular therapies through its ability to resist hypoxia and recruit blood vessels. The growth factors parathyroid hormone related peptide (PTHrP) and indian hedgehog (IHH) are major effectors of GP development via a negative feedback gradient loop. It is unknown if PTHrP and IHH gradients are sufficient to generate GP zonal structure. We hypothesize that counter gradients of PTHrP and IHH agonists across a hydrogel construct seeded with MSC-derived chondrocytes will induce cell differentiation into a GP spatial architecture. We will create a novel model using microfluidic bioreactors and photopatterning of cells and hydrogels. Aim 1 is to design and generate model morphogen gradient profiles in hydrogels. We will generate uniaxial gradients of fluorescently tagged "idealized" morphogens across poly(ethylene glycol) diacrylate (PEGDA) gels housed within our bioreactor. Aim 2 is to investigate growth GP-like zonal differentiation of chondrocytes in generated constructs versus assembled constructs in vitro. We will prepare MSC-derived chondrocytes with culture in supplemented chondrogenic medium. To create generated constructs, we will isolate and embed these chondrocytes in PEGDA gels, and subject gels to a PTHrP and IHH agonist counter-gradient and individual gradients. To create assembled constructs, we will pre-differentiate chondrocytes into reserve and hypertrophic populations with PTHrP and IHH agonist supplemented cultures, and then photopattern these at opposite ends of untreated cells forming a tri-layered gel. Aim 3 is to evaluate engineered GP development and integration with vasculature in vivo. We will implant these constructs in dorsal subcutaneous pockets of immunodeficient mice and use histological and micro-CT assays to evaluate construct growth and GP-like structure maintenance.