PTHrP/IHH Microenvironment Control for Growth Plate Tissue Engineering

PTHrP/IHH 生长板组织工程微环境控制

基本信息

批准号：
8280645
负责人：
Juan Manuel Taboas
金额：
$ 12.57万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8280645
关键词：
15 year old Achievement Agonist Alginates Animal Model Architecture Autologous Transplantation Automobile Driving Biological Assay Biomechanics Bioreactors Blast Injuries Blood Blood Vessels Bone Growth Bone Injury Bone Lengthening Bone Marrow Bone Marrow Stem Cell Bone Regeneration Cartilage Cell Density Cell Differentiation process Cell Proliferation Cells Cellular Morphology Child Chondrocytes Complex Culture Media Deformity Deposition Development Dextrans Differentiation Antigens Distraction Osteogenesis Dorsal Dysplasia Engineering Epiphysial cartilage Erinaceidae Ethylene Glycols External Fixation Devices Feedback Filler Fracture Funding Gel Goals Grant Growth Growth Factor Healed Housing Human Resources Hydrogels Hypertrophy Hypoxia Image Immunodeficient Mouse Implant In Vitro Individual Infection Injury Length Limb structure Maintenance Marrow Measures Mentored Research Scientist Development Award Mesenchymal Stem Cells Microfluidics Modeling Morbidity - disease rate Natural regeneration Osteogenesis Pain Patients Pattern Permeability Population Procedures Radial Recruitment Activity Reporter Research Research Support Rest Risk Signal Pathway Signal Transduction Signaling Molecule Site Skeletal Development Skin Stem cells Structure Testing Time Tissue Engineering Tissues Training Work X-Ray Computed Tomography adult stem cell biomedical scientist bone bone healing bone loss career career development cartilage cell cell type combat design dextran ethylene glycol face bone structure healing in vivo injured limb bone mineralization minimally invasive morphogens multidisciplinary novel parathyroid hormone-related protein poly(ethylene glycol)diacrylate repaired skeletal skeletal disorder skeletal dysplasia skeletal injury subcutaneous tissue regeneration tool

项目摘要

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. PUBLIC HEALTH RELEVANCE: The goal 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 research focus is to create a tissue engineered growth plate model from adult stem cells using cell patterning and controlled growth factor delivery tools. With this novel model, we will be able to study cellular signaling molecules and pathways that cannot be feasibly investigated with animal models and develop treatments to repair congenital skeletal deformities and complex bone injuries that normally heal poorly.

描述（由申请人提供）：这项职业发展赠款的目的是将自己确立为独立的生物医学科学家，他们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。该研究的最终目的是组织生长板（GP）来治疗骨骼发育不良和复杂的骨损伤。 GP是肢体骨头末端的软骨结构，可驱动纵向生长。为了恢复GP患者的肢体长度和几何形状，面部和肢体骨损伤受损，患者通常会使用刺穿皮肤的外部固定器来分心成骨，负担重，长时间和长时间（3-6个月）的手术，引起极大的疼痛，以及风险感染和恐惧。使用骨髓干细胞（MSC）衍生的组织工程GP的骨骼再生和延长可能会比自体移植和细胞疗法通过抵抗缺氧和募集血管的能力更少侵入性和有效性。甲状旁腺激素相关肽（PTHRP）和印度刺猬（IHH）的生长因子是通过负反馈梯度循环的主要效果。未知PTHRP和IHH梯度是否足以生成GP Zonal结构。我们假设PTHRP和IHH激动剂的反梯度在带有MSC衍生的软骨细胞的水凝胶构建体中，将诱导细胞分化为GP空间结构。我们将使用细胞和水凝胶的微流体生物反应器和光结膜创建新型模型。目的1是在水凝胶中设计和生成模型的形态梯度谱。我们将生成跨我们生物反应器中的聚（乙二醇）二丙酸酯（PEGDA）凝胶的荧光标记的“理想化”形态的单轴梯度。 AIM 2是研究软骨细胞的生长GP样区域分化在生成的构建体中与组装构造的体外。我们将在补充软骨培养基中制备具有培养的MSC衍生的软骨细胞。为了创建生成的构建体，我们将将这些软骨细胞隔离在PEGDA凝胶中，并将凝胶凝胶化为PTHRP和IHH激动剂的反梯度和单个梯度。为了创建组装的构建体，我们将用PTHRP和IHH激动剂补充培养物将软骨细胞预分化为储备和肥厚群，然后在未处理的细胞的相对末端拍摄这些培养基，形成三层凝胶。目标3是评估工程的GP开发和与脉管系统的整合体内。我们将将这些构建体植入免疫缺陷小鼠的背皮下袋中，并使用组织学和微CT分析来评估构建体的生长和类似GP的结构维持。公共卫生相关性：这项职业发展的目标是将自己确立为一名独立的生物医学科学家，他们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。研究重点是使用细胞模式和受控生长因子递送工具创建来自成人干细胞的组织工程生长板模型。通过这种新型模型，我们将能够研究细胞信号分子和途径，这些分子和途径无法通过动物模型进行可行研究，并开发治疗以修复通常愈合较差的先天性骨骼畸形和复杂的骨损伤。