Identifying the superior ossification pathway for tissue engineered approaches to long bone repair

项目摘要 肌肉骨骼健康是影响每个人的行动能力和生活质量的关键决定因素， 无论年龄。在美国每年发生的600万例骨折中，高达20%将导致骨不连或 愈合缓慢且需要干预骨再生。间充质基质细胞（MSC）是一种 骨移植物的替代品，因为它们具有成骨、成软骨和促血管生成的潜力。相比 与单分散细胞相比，MSC球状体更好地抵抗细胞凋亡，并分泌100倍高水平的血管生成因子。 同时保持其多能性。然而，MSC球状体还不足以桥接大骨 缺陷，这表明需要有效的编程方法，以提高其骨形成的潜力。 此外，目前还不清楚是通过启动软骨形成或 试图诱导驻留或移植细胞的成骨分化。局部输送诱导 生长因子如BMP-2和TGF-b1加速组织形成，但必要的超生理 浓度和相关并发症损害了它们的广泛临床应用。我们的数据证明 MSC球状体负载细胞分泌的细胞外基质（ECM）对有效的有丝分裂原更敏感 并且在使用显著减少的剂量时表现出增强的成骨和成软骨分化， 减少禁忌症。因此，我们的中心假设是MSC球状体可以在原位分化， 通过呈递吸附到结合的ECM的诱导信号， 这将产生有效的细胞构建块来再生大量丢失的骨体积。目标1.适应细胞- 分泌的ECM在球状体内局部地将诱导因子呈递给MSC，以增强成骨或成纤维细胞增殖。 软骨分化。我们将测试ECM量和BMP-2和TGF-β 1剂量对生长的作用 加载的形态发生素的因子保留和呈递以及它们对骨生成的相关影响 和体外软骨形成。目标二。增强MSC成骨或成软骨分化， 操纵ECM驱动的形态原呈递。我们将确定和量化整合素表达的变化 和含有ECM吸附的BMP-2或TGF-β 1的MSC球体中的生长因子受体活性。我们将 然后评估当分离细胞粘附和生长因子的协同作用时MSC分化的变化， 空房的目标3。确立ECM吸附的外源性形态发生素的治疗潜力， 原位MSC球体用于骨形成。我们将确定含有ECM的MSC球体的能力- 吸附BMP-2或TGF-β 1持续存在，原位进行成骨或成软骨分化，并修复 大面积骨缺损我们将使用非侵入性成像和组织学来描述上级途径， 促进长骨缺损处强健的骨形成。这项研究是创新的，因为它提供了 一种减少引导细胞功能所需的重组生长因子数量的新策略， 确定MSC定向于软骨还是骨在长骨中实现更快的骨修复。

PROJECT SUMMARY Musculoskeletal health is a key determinant of mobility and quality of life which affects every individual, regardless of age. Up to 20% of the 6 million fractures occurring annually in the US will result in nonunion or slow healing and require intervention for bone regeneration. Mesenchymal stromal cells (MSCs) are one alternative to bone grafts because of their osteogenic, chondrogenic, and proangiogenic potential. Compared to monodisperse cells, MSC spheroids better resist apoptosis and secrete 100-fold higher levels of angiogenic factors while retaining their multipotency. However, MSC spheroids are not yet sufficient to bridge large bone defects, suggesting the need for effective programming methods to enhance their bone-forming potential. Furthermore, it is unclear whether bone healing is more effective by jumpstarting cartilage formation or attempting to induce osteogenic differentiation of resident or transplanted cells. Local delivery of inductive growth factors such as BMP-2 and TGF-b1 accelerates tissue formation, but the necessary supraphysiological concentrations and associated complications impair their widespread clinical use. Our data demonstrate that MSC spheroids loaded with cell-secreted extracellular matrix (ECM) are more responsive to potent mitogens and exhibit enhanced osteogenic and chondrogenic differentiation while using markedly reduced dosages and reducing contraindications. Thus, our central hypothesis is that MSC spheroids can be differentiated in situ toward the osteogenic or chondrogenic lineage by presenting inductive cues adsorbed to incorporated ECM, which will yield potent cellular building blocks to regenerate large lost bone volumes. Aim 1. Adapt cell- secreted ECM to locally present inductive factors to MSCs within spheroids to enhance osteogenic or chondrogenic differentiation. We will test the role of ECM quantity and BMP-2 and TGF-b1 dosage on growth factor retention and presentation of loaded morphogens and correlating their resulting effect on osteogenesis and chondrogenesis in vitro. Aim 2. Potentiate MSC osteogenic or chondrogenic differentiation by manipulating ECM-driven morphogen presentation. We will identify and quantify changes in integrin expression and growth factor receptor activity in MSC spheroids containing ECM-adsorbed BMP-2 or TGF-b1. We will then assess changes in MSC differentiation when decoupling the synergy of cell adhesion and growth factor availability. Aim 3. Establish the therapeutic potential of ECM-adsorbed exogenous morphogens to instruct MSC spheroids in situ for bone formation. We will determine the capacity of MSC spheroids containing ECM- adsorbed BMP-2 or TGF-b1 to persist, undergo osteogenic or chondrogenic differentiation in situ, and repair large bone defects. We will use noninvasive imaging and histology to describe the superior pathway to promote robust bone formation in long bone defects. The proposed research is innovative because it provides a novel strategy to reduce the quantity of recombinant growth factors needed to guide cell function, while establishing whether MSCs directed toward cartilage or bone achieve faster bone repair in long bones.