Directed Differentiation of Human Mesenchymal Stem Cells for Bone Repair

描述（由申请人提供）：该项目的长期目标是基于成人间充质干细胞（hMSC）在定义的三维（3D）微环境中的定向分化来创建新的骨移植材料，其可以作为糊剂直接递送到骨修复部位。hMSC是骨组织工程的一种有前途的自体细胞来源，因为它们被证明具有增殖能力，以及清楚地分化成成骨谱系的能力。限制它们在骨修复中使用的主要因素是不能可靠地控制成骨分化的诱导和维持。该项目通过将hMSC包埋在限定的细胞外基质（ECM）和生长因子组成的蛋白质水凝胶珠（直径20 - 200 μ m）中，通过仔细控制hMSC周围的细胞外环境来直接解决这个问题。中心假设是hMSC分化可以通过细胞微环境中的结构和组成变化来指导。结合天然ECM蛋白的特异性整合素和在3D微环境中高效局部递送生长因子的组合预期为hMSC的成骨分化提供有力的刺激。该项目的具体目标是：1]鉴定一致地促进hMSC向成骨表型分化的3D细胞外微环境制剂，2]用BMP-2（已知具有有效骨诱导作用的生长因子）增加细胞外微环境，3]使限定的细胞微环境的使用适应于高密度培养，并将它们作为可注射的细胞递送系统在体外进行测试，和4]在免疫缺陷大鼠的节段性缺损模型中体内测试3D基质包埋的hMSC。创建定义的3D微珠微环境的方法解决了对有效骨修复至关重要的四个关键要素：i]活成骨细胞（hMSC）的存在，ii]骨诱导支架（天然ECM蛋白胶原I和玻连蛋白），iii）向驻留细胞提供信号的骨诱导生长因子（BMP-2），和iv]足够的血液供应以支持细胞生长和功能（通过在珠粒之间提供空隙空间用于体内血管向内生长和VEGF递送）。该项目旨在解决临床问题，这些问题可以从在困难环境中加速骨愈合的治疗中受益，例如缺血性坏死，脊柱融合和植入物固定。它将产生hMSC表型如何被3D细胞外环境控制的基础知识，因此将对再生医学产生广泛的影响。在骨科组织工程领域，它将产生一种新的基于细胞的骨修复疗法。

DESCRIPTION (provided by applicant): The long-term objective of this project is to create new bone graft materials based on the directed differentiation of adult human mesenchymal stem cells (hMSC) in defined three-dimensional (3D) microenvironments, which can be delivered as a paste directly to the site of bone repair. hMSC are a promising autogenous cell source for bone tissue engineering because of their demonstrated ability to proliferate, as well as to clearly differentiate into the osteogenic lineage. A main factor limiting their use in bone repair is the inability to reliably control the induction and maintenance of osteogenic differentiation. This project directly addresses this issue by carefully controlling the extracellular environment immediately surrounding hMSC, by embedding them in protein hydrogel beads (20-200 um in diameter) of defined extracellular matrix (ECM) and growth factor composition. The central hypothesis is that hMSC differentiation can be directed through structural and compositional changes in the cellular microenvironment. The combination of specific integrin binding to natural ECM proteins and highly efficient, local delivery of growth factors in a 3D microenvironment is expected to provide a potent stimulus for osteogenic differentiation of hMSC. The Specific Aims of this project are to: 1] identify 3D extracellular microenvironment formulations that consistently promote differentiation of hMSC towards the osteogenic phenotype, 2] augment the extracellular microenvironment with BMP-2, a growth factor known to have potent osteoinductive effects, 3] adapt the use of defined cellular microenvironments to high density culture, and test them as an injectable cell delivery system in vitro, and 4] test 3D matrix-embedded hMSC in vivo in a segmental defect model in immunodeficient rats. The approach of creating defined 3D bead microenvironments addresses four key elements that are critical to effective bone repair: i] the presence of living osteogenic cells (hMSC), ii] an osteoinductive scaffold (the natural ECM proteins collagen I and vitronectin), iii] osteoinductive growth factors to provide signals to the resident cells (BMP-2), and iv] an adequate blood supply to support cell growth and function (by providing void spaces between beads for vascular ingrowth and VEGF delivery in vivo). This project is aimed at clinical problems that can benefit from therapies that accelerate bone healing in a difficult environment, such as avascular necrosis, spinal fusion and implant fixation. It will yield fundamental knowledge of how hMSC phenotype can be controlled by the 3D extracellular environment, and will therefore have broad impact on regenerative medicine. In the field of orthopaedic tissue engineering, it will yield a new cell-based bone repair therapy.