Tensile Strain-Induced Osteogenesis of Human Mesenchymal Stem Cells in 3D Culture

3D 培养中拉伸应变诱导人间充质干细胞成骨

• 批准号: 7789382
• 负责人: Elizabeth Grace Loboa
• 金额: $ 7.24万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7789382
• 关键词: Adipose tissue; Adverse reactions; Alkaline Phosphatase; Area; Attention; Autologous Transplantation; Biochemical; Bone Tissue; Bone Transplantation; Calcium; Cell Line; Cells; Chemicals; Chondrogenesis; Cilia; Collagen; Culture Media; Data; Defect; Deposition; Electroplating; Environment; Exhibits; Failure; Fatty acid glycerol esters; Future; Gel; Genes; Genetic; Gold; Hair; Harvest; Human; Infection; Lead; Mature Bone; Mechanics; Mesenchymal Stem Cells; Methacrylates; Minerals; Operative Surgical Procedures; Organelles; Osteogenesis; Pain; Patients; Physical Stimulation; Play; Population; Procedures; Property; Public Health; Relative (related person); Replacement Therapy; Role; Site; Small Interfering RNA; Source; Stem cells; Stimulus; Structure; Testing; Tissue Engineering; Tissues; Transplanted tissue; Weight-Bearing state; Work; appendage; base; bone; bone cell; human stem cells; in vivo; irritation; lipid biosynthesis; myogenesis; osteogenic; public health relevance; reconstruction; response; sensor; stem cell fate; wound

DESCRIPTION (provided by applicant): Bone is the most transplanted tissue. The gold standard for bone grafts (90% of surgeries) is the autograft. Limitations of this procedure include a limited supply of bone in the body, infection, and pain at the donor site. Therefore, cell-based strategies have received much attention as a replacement therapy, with particular focus on functional tissue engineering. Functional tissue engineering uses physical stimulation to direct cell populations to produce tissue with anatomically and physiologically correct structures and material properties similar to native tissue. Adipose-derived stem cells (ASCs) are a particularly promising cell source for functional tissue engineering applications due to their multilineage differentiation potential and their relative abundance and ease of harvest relative to many other cell lines. However, there is a dearth of information on the mechanobiology of human ASCs (hASCs). Mechanical loads that promote osteodifferentiation of hASCs and creation of functional bone are unknown, as are the mechanotransduction mechanisms associated with load-induced hASC osteodifferentiation. It has been recently shown in bone cells, however, that the primary cilium, a hair-like appendage that exists on almost all cells, plays a crucial mechanotransduction role during bone formation. We have collected unique preliminary data showing that cyclic tensile strain of magnitude 10% accelerates hASC osteodifferentiation and significantly increases calcium accretion. We have also found that hASCs possess a primary cilium, an organelle in bone cells recently shown to play a vital mechanotransduction role during bone formation. Overall Hypothesis: Primary cilia function as sensors of cyclic tensile strain in 3D culture and thereby regulate hASC osteogenesis and functional bone formation. Specific Hypotheses and Objectives/Aims: 1. Primary cilia are responsible, in whole or in part, for transduction of cyclic tensile strain to hASCs in 3D culture. Determine the role of primary cilia in the transduction of cyclic tensile strain to hASCs in 3D culture. Human ASCs will be cultured in 3D collagen gels in both the presence and absence of osteogenic supplements in the culture medium and exposed to 10% cyclic tensile strain - a magnitude we have recently determined to induce and accelerate osteogenesis and increase calcium accretion of hASCs. Primary cilia will be inhibited both biochemically and with a siRNA knockdown strategy and the osteodifferentiation response of hASCs primary cilia characterized. 2. In the absence of primary cilia, bone formed by hASCs will exhibit reduced stiffness and strength. Determine the role of hASC primary cilia in functional bone tissue engineering. Primary cilia will be inhibited as described in SA1. Human ASCs primary cilia will be cultured in 3D collagen gels and exposed to biochemical and mechanical stimuli to induce hASC osteodifferentiation and bone formation. Bone constructs will be tested to failure in tension and compression. Material properties of bone constructs formed by hASCs without primary cilia will be compared to those of bone generated by hASCs with primary cilia and to mature bone. Impact: While it is now understood that mechanical loading is a requirement for successful tissue engineering of load-bearing tissues, it is not known how loading controls stem cell fate or is transduced at the cellular level. Determining the mechanical environment that causes hASC osteodifferentiation and functional bone formation and how those loads are transduced at the cellular level will allow us to optimize hASC response using a synergistic combination of both mechanical and biochemical environments. If it is determined that primary cilia play a vital mechanotransduction role in functional bone formation by hASCs, future work can investigate biochemical or genetic means to induce greater expression of primary cilia in a population of hASCs and/or lengthen hASC primary cilia. Longer primary cilia would be more mechanosensitive (e.g., less load required to bend the primary cilia), allowing for formation of structurally and mechanically robust bone (this is not presently attained by chemical osteogenic supplements alone) by hASCs, even in environments of reduced mechanical load. Such approaches would lead to patient-specific functional bone formation, using a patient's own fat-derived stem cells, and creation of tissue engineered bone capable of withstanding in vivo loads. PUBLIC HEALTH RELEVANCE: The gold standard for bone grafts (90% of surgeries) is the autograft where bone is removed from one portion of the patient's body and grafted to a wound elsewhere. While minimizing tissue rejection, limitations of this procedure include limited supply of bone in the body, infection, and pain at the donor site. Large bone defects can be reconstructed with materials such as metal plates or methyl methacrylate. However, these are foreign materials and may cause a variety of adverse reactions, including irritation, encapsulation of the replacement material, and eventual exposure of the reconstructed area. If this happens, the foreign material has to be removed and the site reconstructed with bone. Bone truly is the best material for reconstruction of bone defects. Being able to create a tissue- engineered bone construct using a patient's own stem cells would eliminate the need for painful bone grafts and/or use of foreign materials to fill critical-sized bone defects.

描述（由申请人提供）：骨骼是最移植的组织。骨移植的金标准（手术的90％）是自体移植。此过程的局限性包括体内骨骼的有限供应，感染和供体部位的疼痛。因此，基于细胞的策略已被广泛关注作为一种替代疗法，特别关注功能组织工程。功能组织工程使用物理刺激来指导细胞群，以解剖和生理上正确的结构和材料特性类似于天然组织。脂肪来源的干细胞（ASC）是功能组织工程应用的特别有希望的细胞来源，因为它们的多限性分化潜力及其相对丰度和相对于许多其他细胞系的相对丰度和易于收获。但是，缺乏有关人类ASC机械生物学（HASC）的信息。促进HASC和功能骨创建的骨化阶段的机械载荷是未知的，与负载诱导的HASC Osteodfferentiation相关的机械传输机制也是未知的。然而，最近在骨细胞中表明，几乎所有细胞上存在类似头发的附属物的原发性纤毛在骨形成过程中起着至关重要的机械转移作用。我们已经收集了独特的初步数据，表明循环拉伸应力为10％加速度的hasc骨化分化，并显着增加钙的积聚。我们还发现，HASC具有原发性纤毛，这是骨细胞中最近显示的骨细胞中的细胞器，在骨形成过程中起着至关重要的机械转导作用。总体假设：原发性纤毛在3D培养中充当环状拉伸应变的传感器，从而调节HASC成骨的发生和功能性骨形成。特定的假设和目标/目的：1。主要纤毛全部或部分原因是将环状拉伸应变转导向3D培养中的HASC。确定纤毛在3D培养中循环拉伸应变向HASC的转导中的作用。在培养基中的成骨补充剂的存在和不存在的情况下，将在3D胶原蛋白凝胶中培养人类ASC，并暴露于10％的环状拉伸应变 - 我们最近确定的幅度是诱导和加速成骨的作用并增加HASC的钙增生。原发性纤毛将在生物化学和siRNA敲低策略和hascs原发性纤毛的骨化反应反应上均受到抑制。 2。在没有原发性纤毛的情况下，由HASC形成的骨骼将表现出降低的刚度和强度。 确定HASC一级纤毛在功能性骨组织工程中的作用。如SA1中所述，将抑制原发性纤毛。人类ASC的原发性纤毛将在3D胶原蛋白凝胶中培养，并暴露于生化和机械刺激中，以诱导Hasc Osteationdferentiation和骨形成。骨骼构建体将在张力和压缩中测试。将没有原发性纤毛的HASC形成的骨骼构建体的材料特性将与原代纤毛和成熟骨骼产生的骨骼的骨骼进行比较。影响：虽然现在已经理解，机械负荷是成功的载荷组织组织工程的必要条件，但尚不清楚加载如何控制干细胞命运或在细胞水平上转导。确定导致HADC骨化分化和功能性骨形成的机械环境以及在细胞水平下如何转导的负载将使我们能够使用机械和生化环境的协同组合来优化HASC响应。如果确定原发性纤毛在HASC中起着至关重要的机械转导作用，则未来的工作可以研究生化或遗传方法，以诱导HASC人群中纤毛的更大表达和/或延长HASC原发性纤毛。较长的原发性纤毛更具机械性敏感性（例如，弯曲原发性纤毛所需的负载较小），即使在机械负载降低的环境中，也可以在结构和机械上稳健的骨骼形成（目前无法通过化学成骨的骨骼补充剂来实现）。这种方法将使用患者自身的脂肪衍生的干细胞以及能够承受体内负荷的组织工程骨的创建，从而导致患者特异性的功能性骨形成。 公共卫生相关性：骨移植的黄金标准（手术的90％）是自体移植，其中从患者身体的一部分中去除骨骼并将其移植到其他地方的伤口。在最大程度地减少组织排斥反应的同时，此过程的局限性包括体内骨骼的供应有限，感染和供体部位的疼痛。可以用金属板或甲基丙烯酸甲酯等材料重建大骨缺陷。但是，这些是异物，可能会引起多种不良反应，包括刺激，替代材料的封装以及最终暴露于重建区域。如果发生这种情况，则必须去除异物，并用骨头重建现场。骨骼确实是重建骨缺损的最佳材料。能够使用患者自己的干细胞创建组织骨骼构建体，这将消除对疼痛骨移植的需求和/或使用异物来填补关键大小的骨缺损。