PTHrP/IHH Microenvironment Control for Growth Plate Tissue Engineering

描述（由申请人提供）：这个职业发展补助金的目的是建立自己作为一个独立的生物医学科学家谁开发组织再生疗法，通过骨骼发育和疾病的工程微环境模型的研究。该研究的最终目标是组织工程生长板（GP）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，驱动纵向生长。为了恢复患有GP和受损面部和四肢骨损伤的患者的肢体长度和几何形状，患者通常经受牵引成骨，这是一种使用外部固定器的繁重且长时间（3-6个月）的手术，其刺穿皮肤，引起巨大疼痛，并有感染和瘢痕的风险。使用骨髓干细胞（MSC）衍生的组织工程GP的骨再生和延长可能被证明比自体移植物和脱细胞疗法具有更低的侵入性和更有效的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）通过负反馈梯度环是GP发展的主要效应因子。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MSC衍生的软骨细胞的水凝胶结构中的反向梯度将诱导细胞分化为GP空间结构。我们将创建一个新的模型，使用微流体生物反应器和细胞和水凝胶的微图案化。目的1是设计和生成水凝胶中的模型形态基元梯度分布。我们将产生单轴梯度的荧光标记的“理想化”形态发生剂在聚（乙二醇）二丙烯酸酯（PEGDA）凝胶内安置在我们的生物反应器。目的2：探讨软骨细胞生长GP样带状分化 在体外生成的构建体与组装的构建体中。我们将在补充的成软骨培养基中培养MSC衍生的软骨细胞。为了创建生成的构建体，我们将分离这些软骨细胞并将其包埋在PEGDA凝胶中，并使凝胶经受PTHrP和IHH激动剂反梯度和单独梯度。为了创建组装的构建体，我们将用PTHrP和IHH激动剂补充的培养物将软骨细胞预分化成储备和肥大群体，然后在未处理细胞的相对端将这些预分化成图案，形成三层凝胶。目的3是评价工程GP在体内的发育和与血管的整合。我们将这些构建体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测定来评估构建体的生长和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.