PTHrP/IHH Microenvironment Control for Growth Plate Tissue Engineering

描述（由申请人提供）：这项职业发展资助的目的是使自己成为一名独立的生物医学科学家，通过研究工程微环境模型中的骨骼发育和疾病来开发组织再生疗法。该研究的最终目标是组织工程生长板（GP）来治疗骨骼发育不良和复杂的骨损伤。GP是肢体末端的软骨结构，驱动纵向生长。为了恢复GP和面部及四肢骨损伤患者的肢体长度和几何形状，患者通常需要牵张成骨术，这是一个负担沉重且耗时（3-6个月）的手术，使用外固定架刺穿皮肤，引起巨大疼痛，并有感染和恐吓的风险。使用骨髓干细胞（MSC）衍生的组织工程GP进行骨再生和延长可能比自体移植和脱细胞治疗侵入性更小，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hedgehog基因（IHH）是通过负反馈梯度回路影响GP发育的主要因子。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激动剂在水凝胶构建体中的反梯度与msc衍生软骨细胞的种子将诱导细胞分化成GP空间结构。我们将使用微流控生物反应器和细胞和水凝胶的光图像化来创建一个新的模型。目的1是设计和生成水凝胶中形态发生梯度的模型。我们将在生物反应器内的聚乙二醇二丙烯酸酯（PEGDA）凝胶上产生荧光标记的“理想化”形态素的单轴梯度。目的2研究软骨细胞生长gp样的区向分化

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.