CAREER: Tissue engineering hematopoietic trabecular bone marrow

每天，人体的成体干细胞库，被称为造血干细胞（hsc），发育成近一万亿成熟血细胞，包括红细胞、白细胞、血小板和免疫细胞。这些造血干细胞主要存在于小梁骨髓（TBM）中，这是长骨（如股骨）多孔末端的组织。造血干细胞移植是最成功的干细胞治疗方法，但其临床效果受到造血干细胞可用性的限制。HSC很难在体外生长，因为它们往往会发生变化（分化），并失去发展成任何血细胞类型的能力。为了满足对可扩展的HSC生长方法的需求，而不允许分化，本CAREER项目的目标是开发模拟TBM微环境的生物材料模型，用于HSC的生长和维持。一旦开发成功，这些生物材料模型将被结合并集成到设计用于培养造血干细胞的生物反应器中的TBM模型中。成功的TBM模型将促进对与衰老和骨质疏松症和骨癌等疾病进展相关的骨重塑的理解，并且它们也可以在临床前药物试验中替代动物。此外，该项目将用于招收和教育具有不同背景的学生，以面对工程和医学交叉领域的新挑战。活动包括组织工程课程的综合讲座和实验课程，面向本科生的跨学科团队（工程和生物学）顶点项目，以及面向高中女生的暑期研究项目，该项目与现有的“细胞工程”项目相结合。研究者的长期研究目标是提供转化生物工程解决方案，以促进我们对健康和疾病中的小梁骨髓（TBM）的理解，并利用TBM的再生潜力。为了实现这一目标，本CAREER项目的目的是阐明维持TBM中造血活性的动态结构-功能关系，并应用这一知识设计可扩展的生物反应器来扩增造血干细胞（hsc）。两个观察结果：(1)造血造血干细胞主要存在于保持相当骨厚度和腔直径的腔中并发挥作用，同时进行持续的骨重塑；(2)随着年龄的增长，小梁骨厚度的减少和腔直径的增加与造血活性的下降有关，这表明在TBM中，解剖结构和维持造血活性的功能之间可能存在关系。因此，该项目的中心假设是存在最佳的尺寸和空间安排，可以有效地协调骨内膜壁龛和血管壁龛之间的双向串扰，这是TBM中两个重要的解剖和功能壁龛。该项目建立在研究者开发的(1)倒置胶体晶体（ICC）水凝胶支架的基础上，该支架密切模仿血管生态位的解剖和物理特征；(2)脱矿骨纸，保留完整的骨内生态位的生化和结构方面，并在复制骨的表面和表面下骨组织复杂性方面显示出生物学意义，包括骨细胞。研究计划有四个目标：(1)通过完善骨内生态位模型，在骨内生态位模型中，骨细胞在机械培养下被埋在脱矿骨纸层中，重建骨表面和亚表面的生理方面，建立一个概括骨重塑相关HSC生物学的骨内生态位模型；(2)通过完善血管生态位模型，模拟骨髓基质细胞（BMSCs）培养的微流控水凝胶支架内灌注下的分子梯度，开发模拟血管灌注相关HSC生物学的血管生态位模型；(3)通过将单独优化的模型整合到一个平台上，将内膜和血管生态位模型整合成一个集成的TBM模型，以重新创建内膜生态位的分化抑制质量和血管生态位的增殖刺激质量。(4)将多个TBM模型组装成一个可扩展的生物反应器，以维持内膜和血管生态位之间稳定的分子和机械相互作用水平，以支持扩张造血干细胞阶段。为了确定体外HSC扩增生物反应器与其他溶液相比的竞争优势和劣势，将进行极限稀释试验，以确定静脉注射亚致死照射小鼠后培养扩增的HSC在体内的再生潜力。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Every day, the human body’s pool of adult stem cells, termed hematopoietic stem cells (HSCs), develop into nearly one trillion mature blood cells, including red blood cells, white blood cells, platelets and immune cells. These blood cell-forming HSCs primarily reside in trabecular bone marrow (TBM), which is the tissue in the porous end of long bones such as femurs. HSC transplantation is the most successful stem cell therapy, but its clinical impact has been limited by the availability of HSCs. HSC’s are difficult to grow outside the body where they tend to change (differentiate) and lose their ability to develop into any blood cell type. To address the need for a scalable method to grow HSCs without allowing differentiation, the goal of this CAREER project is to develop biomaterial models that mimic the microenvironments of TBM for HSC growth and maintenance. Once developed, the biomaterial models will be combined and integrated into a TBM model in bioreactor designed to growth HSCs. Successful TBM models will advance understanding of bone remodeling associated with aging and the progression of diseases such as osteoporosis and bone cancers, and they may also serve as an alternative to animals during preclinical drug testing. Furthermore, the project will be used to recruit and educate students with diverse backgrounds to face emerging challenges at the intersection between engineering and medicine. Activities include an integrated lecture and lab curriculum for a tissue engineering course, an interdisciplinary team-based (engineering and biology) capstone projects for undergraduate students, and a summer research program for high school girls aligned with the existing Engineering the Cell program.The investigator’s long-term research goals are to deliver translational bioengineered solutions that can advance our understanding of the trabecular bone marrow (TBM) in health and disease and harness the regenerative potential of the TBM. Toward this goal, the aim of this CAREER project is to elucidate the dynamic structure-function relationship that maintains hematopoietic activity in the TBM and to apply this knowledge to design a scalable bioreactor for expanding hematopoietic stem cells (HSCs). Two observations, (1) that blood-forming HSCs primarily reside and function in cavities that maintain comparable bone thickness and cavity diameters while undergoing constant bone remodeling and (2) that aging-associated decrease in trabecular bone thickness and increase in cavity diameters are related to a decrease in hematopoietic activity, suggest a possible relationship between anatomy and function that maintains hematopoietic activity in the TBM. Thus, the project’s central hypothesis is that there exist optimal dimensions and spatial arrangement that effectively coordinate bidirectional crosstalk between the endosteal and vascular niches, which are the two important anatomical and functional niches in the TBM. The project builds on the investigator’s development of (1) inverted colloidal crystal (ICC) hydrogel scaffolds that closely emulate anatomical and physical features of the vascular niche and (2) demineralized bone paper that preserves intact biochemical and structural aspects of the endosteal niche and exhibits biological significance in reproducing the surface and subsurface bone tissue complexity of bone, including osteocytes. The Research Plan is organized under four Objectives: (1) To develop an endosteal niche model that recapitulates bone remodeling-related HSC biology by refining an endosteal niche model in which osteocytes buried in layers of demineralized bone paper under mechanoculture re-create physiological aspects of bone surface and subsurface; (2) To develop a vascular niche model that emulates vascular perfusion-related HSC biology by refining the model of the vascular niche to mimic molecular gradients under perfusion within a microfluidic hydrogel scaffold that supports culture of bone marrow stromal cells (BMSCs); (3) To combine the endosteal and vascular niche models into an integrated TBM model by integrating individually optimized models into a single platform to re-create the differentiation-suppressing quality of the endosteal niche and the proliferation-stimulating quality of the vascular niche and (4) To assemble multiple TBM models into a scalable bioreactor that will maintain a stable level of molecular and mechanical interactions between the endosteal and vascular niches to support the expansion phase of HSCs. To determine the competitive advantages and weaknesses of the ex vivo HSC expansion bioreactor compared to other solutions, a limiting dilution assay will be conducted to determine the in vivo repopulating potential of the culture-expanded HSCs by intravenous injection into sublethally irradiated mice.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.