外周静脉移植间质干细胞对骨创伤修复作用的机制研究及其影像学评价

Although intravenous-mesenchymal stem cell (IV-MSC) therapy has great potential for tissue repair and regeneration for various indications, including fracture insufficiency and non-unions in the elderly, several aspects of its clinical application warrant significant improvement. Most notably, an adjuvant therapy that can: 1) enhance MSC proliferative potential prior to differentiation at the injury, 2) improve MSC targeting to the damaged tissue, and 3) sustain MSC pluripotency for differentiation into all the cell types needed to heal the injury. Additionally, while genetic marker (i.e. LacZ, GFP) experiments can be used to address some of these questions in animal models, these modified cells cannot be used in people. Thus, there is a need for translational approaches to evaluate IV-MSC therapies in patients, such as MRI tracking of superparamagnetic iron oxide labeled cells. Towards these goals, we aim to evaluate the effects of PTH (teriparatide) on human IV-MSC therapy in a murine femur fracture model, in which in vivo targeting is assessed by novel MRI detection of ex vivo fluorescent iron oxide nanoparticle (IODEX-TAT-FITC) labeled hMSC, and confirmed by histology. Unfortunately, we have also found that this ex vivo labeling procedure results in the loss of hMSC "stemness" as evidenced by a dramatic loss of stem cell gene expression (nanog, oct-4, sox-2), and the lack of osteogenic differentiation of these labeled hMSC at the fracture site in vivo, which limits the therapeutic effect of IV-MSC therapy over PTH therapy alone. Thus, here we propose to overcome this limitation by reprogramming the Fe++labeled hMSC in vitro with PTH treatment prior to IV-MSC therapy, and testing an alternative ex vivo 19-F perfluorocarbon polymer labeling reagent as follows. In Aim 1: we will demonstrate the effects of culturing (1) IODEX-TAT-FITC (Fe++-ITF) particle or (2) Cell Sense-FITC (CSF) labeled hMSC in PTH on their stemness and pluripotency in vitro and in vivo. The conditions that produce the hMSC phenotype that most closely resembles unlabeled hMSC controls will be evaluated in our murine model of IV-MSC +/- PTH fracture healing model in which in vivo homing to the fracture callus will be assessed by longitudinal MRI, and confirmed by Prussian blue stained histology. In Aim 2: we will determine the adjuvant effects of PTH on IV-hMSCs therapy on fracture callus in elderly mice. We will evaluate the effects of: Group 1) placebo, 2) standard cultured IV-hMSC, 3) PTH cultured IV-hMSC, 4) PTH therapy, 5) standard cultured IV-MSC + PTH therapy, and 6) PTH cultured IV-MSC + PTH therapy in our aging fracture model using longitudinal MRI detection of Fe++-ITF or CSF labeled hMSC, and fracture healing by micro-CT, biomechanical testing, and histology.If successful, the results from this work will be used to justify a novel clinical trial of IV-MSC + PTH therapy in geriatric patients with inoperable fracture insufficiency.

间质干细胞（hMSC）是促进骨折修复最重要因素,但目前其移植后的增殖、靶向迁移与归巢、定向分化及疗效评价等问题尚未解决。申请者现有的动物模型及LacZ/GFP标记的hMSC虽可部分回答上述问题，但临床应用仍有局限。因此，探索可转化应用的hMSC移植方法、示踪其归巢及分化潜能、疗效评价等成为迫切需要。本项目拟用超顺磁性纳米颗粒（ITF/19-F)与LacZ/GFP干细胞标记、Micro-CT和3T-MRI特制小鼠高分辨率线圈及扫描、甲状旁腺素（rhPTH)辅助的外周静脉干细胞移植等技术结合研究，以明确1）rhPTH对ITF/19-F标记的hMSC的增殖能力、定向分化成骨的效能；2）治疗剂量的hPTH改善hMSC靶向迁移-归巢的微环境，促进骨折癒合改善骨生物力学特性。提供直接证据、完善hMSC临床转化理论基础；优化hMSC移植量-效条件；提供3-T MRI示踪hMSC的技术参数。

间质干细胞（hMSC）是促进骨折修复最重要因素，但目前其移植后的增殖、靶向迁移与归巢、定向分化及疗效评价等问题尚未解决。申请者运用动物模型及LacZ/GFP标记的hMSC明确回答部分上述问题外，还探索了可转化应用的hMSC移植方法、示踪其归巢及分化潜能、疗效评价等。本项使用超顺磁性纳米颗粒（ITF/19-F)与LacZ/GFP干细胞标记、Micro-CT和3T-MRI特制小鼠高分辨率线圈及扫描、甲状旁腺素（rhPTH)辅助的外周静脉干细胞移植等技术结合研究，明确了1）rhPTH对ITF/19-F标记的hMSC的增殖能力、定向分化成骨的效能；2）治疗剂量的hPTH改善hMSC靶向迁移-归巢的微环境，促进骨折癒合改善骨生物力学特性。提供了直接证据、完善hMSC临床转化理论基础；优化了hMSC移植量-效条件；提供了3-T MRI示踪hMSC的技术参数。并拓展性延伸MSC在脊柱脊髓损伤及恶性肿瘤导致骨缺损治疗过程的血管定向分化及损伤微环境修复的作用。