Directed Differentiation of Human Mesenchymal Stem Cells for Bone Repair

描述(申请人提供)：该项目的长期目标是创造新的骨移植材料，基于成人间充质干细胞(HMSC)在特定的三维(3D)微环境中的定向分化，可以糊状物的形式直接输送到骨修复部位。人骨髓间充质干细胞具有良好的增殖能力和明显的成骨分化能力，是骨组织工程中一种很有前途的自体细胞来源。限制其在骨修复中应用的一个主要因素是无法可靠地控制成骨分化的诱导和维持。该项目直接解决这一问题，方法是仔细控制hMSC周围的细胞外环境，将它们嵌入到定义了细胞外基质(ECM)和生长因子组成的蛋白质水凝胶微珠(直径20-200微米)中。中心假说是，hMSC的分化可以通过细胞微环境的结构和成分变化来引导。结合天然ECM蛋白的特异性整合素和在三维微环境中高效、局部输送生长因子，有望为hMSC的成骨分化提供强有力的刺激。该项目的具体目的是：1)确定持续促进hMSC向成骨表型分化的3D细胞外微环境配方；2)用BMP-2(已知具有强大的成骨诱导作用的生长因子)增强细胞外微环境；3)使已定义的细胞微环境适用于高密度培养，并测试其作为可注射细胞载体的体外释放系统；4)测试3D基质包埋的hMSC在体内免疫缺陷大鼠节段性缺陷模型中的作用。创建定义的3D微环境的方法解决了对有效的骨修复至关重要的四个关键要素：i)活的成骨细胞(HMSC)的存在，ii)骨诱导支架(天然的ECM蛋白I型胶原和玻璃体连接蛋白)，iii)骨诱导生长因子，向驻留细胞提供信号(BMP-2)，以及iv)足够的血液供应支持细胞的生长和功能(通过在体内提供微珠之间的空隙用于血管内生长和VEGF的输送)。该项目旨在解决临床问题，这些问题可以受益于在困难环境下加速骨愈合的治疗方法，如缺血性坏死、脊柱融合和植入物固定。它将产生关于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.