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）：这项职业发展补助金的目的是使我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通过组织工程设计生长板（GP）来治疗骨骼发育不良和复杂的骨损伤。 GP 是四肢骨末端的软骨结构，驱动纵向生长。为了恢复 GP 和面部和四肢骨损伤受损患者的肢体长度和几何形状，患者通常会接受牵引成骨术，这是一种繁琐且耗时（3-6 个月）的手术，使用的外固定器会刺穿皮肤，造成极大的疼痛，并且存在感染和惊吓的风险。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会证明，由于其具有抵抗缺氧和募集血管的能力，比自体移植和无细胞疗法侵入性更小，更有效。生长因子甲状旁腺激素相关肽 (PTHrP) 和印度刺猬蛋白 (IHH) 通过负反馈梯度循环是 GP 发育的主要效应因子。目前尚不清楚 PTHrP 和 IHH 梯度是否足以生成 GP 区域结构。我们假设，在接种 MSC 衍生软骨细胞的水凝胶结构中，PTHrP 和 IHH 激动剂的反梯度将诱导细胞分化为 GP 空间结构。我们将使用微流体生物反应器以及细胞和水凝胶的光图案化创建一个新颖的模型。目标 1 是设计和生成水凝胶中的模型形态发生素梯度分布。我们将在生物反应器内的聚乙二醇二丙烯酸酯 (PEGDA) 凝胶上生成荧光标记的“理想化”形态发生素的单轴梯度。目标 2 是研究软骨细胞的生长 GP 样区域分化生成的构建体与体外组装的构建体。我们将在补充的软骨形成培养基中培养来制备 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嵌入 PEGDA 凝胶中，并对凝胶进行 PTHrP 和 IHH 激动剂反梯度和单独梯度。为了创建组装的构建体，我们将使用 PTHrP 和 IHH 激动剂补充培养物将软骨细胞预分化为储备细胞和肥大细胞群，然后在未处理细胞的两端对这些细胞进行光图案化，形成三层凝胶。目标 3 是评估工程化 GP 的开发以及与体内脉管系统的整合。我们将这些构建体植入免疫缺陷小鼠的背侧皮下袋中，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。公共健康相关性：这项职业发展补助金的目标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。研究重点是使用细胞模式和受控生长因子递送工具从成体干细胞创建组织工程生长板模型。通过这种新模型，我们将能够研究无法通过动物模型进行可行研究的细胞信号分子和通路，并开发修复通常愈合不良的先天性骨骼畸形和复杂骨损伤的治疗方法。