Human Embryonic Stem Cells in Calcium Phosphate Constructs for Bone Regeneration

描述（由申请人提供）：美国每年有700万人患骨折。肌肉骨骼疾病每年给美国造成的损失超过2000亿美元。随着人口老龄化，这些数字正在迅速增长。人类胚胎干细胞（hESCs）提供了无限的干细胞供应，具有很高的骨再生潜力。然而，目前还没有关于hESCs通过注射磷酸钙用于骨组织工程的报道。虽然人类骨髓间充质干细胞（hBMSCs）是有用的，但它们的获取需要侵入性的过程，并且由于衰老和疾病，它们的增殖和分化潜力很大。因此，本项目的目标是：(1)研究用于骨工程的可注射磷酸钙水泥（CPC）中的hESCs，并与人脐带间充质干细胞（hUCMSCs）和hBMSCs进行比较；(2)建立首个hESC与CPC支架相互作用的知识库，指导hESC的增殖和骨分化；(3)设计rgd移植CPC支架进行hESC包埋，促进骨再生。虽然Arg-Gly-Asp （RGD）肽已用于其他支架，但尚未见其在CPC中的应用报道。目的1将研究hESC在可注射和大孔CPC- rgd结构中的体外包埋和分化，并验证以下假设：(1)hESC来源的MSCs包埋在可降解微珠中并掺入CPC中合成骨基质的量最多，其次是hUCMSCs。金标准hBMSCs将产生最少的骨基质；(2) rgd移植的CPC在不影响CPC可注射性和力学性能的情况下，可大大增强细胞功能和骨基质合成，与报道的松质骨强度相匹配。目的2将研究hESC-CPC-RGD构建的骨再生动物模型，并验证以下假设：(1)使用hesc来源的MSCs， CPC-RGD生成的新骨体积、矿物质密度和血管密度最高，其次是hUCMSCs。两者都将远远超过hBMSCs产生的；(2) hESC-CPC- RGD将在6个月时在整个临界尺寸的颅骨缺损上完全被新骨替代；(3) hESC-CPC-RGD诱导的新骨数量明显高于未进行RGD的小鼠。该项目将在hESC在CPC中的封装、hESC与RGD-CPC的相互作用以及hESCs在比较hESCs、hUCMSCs和hBMSCs的动物模型中对骨分化和骨再生的指导方面获得突破性的知识。这种新型干细胞膏状体可用于微创手术，填补形状复杂的缺陷，并可在牙科和颅面外科应用中易于整形。新型hESC- CPC-RGD结构有望在骨科和颅面外科广泛应用，极大地促进骨再生，改善数百万人的健康和生活质量。如果通过CPC输送的hESCs在成骨方面确实优于金标准的hBMSCs，预计该项目将首次证明这一点，这一结果将广泛影响再生医学

DESCRIPTION (provided by applicant): Seven million people suffer bone fractures in the U.S. each year. Musculoskeletal conditions cost the U.S. more than $200 billion annually. These numbers are increasing rapidly as the population ages. Human embryonic stem cells (hESCs) offer unlimited supplies of stem cells with a high potential for bone regeneration. However, there has been no report on the use of hESCs for bone tissue engineering via injectable calcium phosphates. While human bone marrow mesenchymal stem cells (hBMSCs) are useful, their harvest requires an invasive procedure, and their proliferation and differentiation potential is lot due to aging and diseases. Therefore, the objectives of this project are to: (1) investigate hESCs in injectable calcium phosphate cement (CPC) for bone engineering, in comparison with human umbilical cord MSCs (hUCMSCs) and hBMSCs; (2) establish the first knowledge base on hESC interactions with CPC scaffolds to guide hESCs for proliferation and osteodifferentiation; (3) design a RGD-grafted CPC scaffold for hESC encapsulation to enhance bone regeneration. While the Arg-Gly-Asp (RGD) peptide has been used in other scaffolds, there has been no report on its use in CPC. Aim 1 will investigate in vitro the hESC encapsulation and differentiation in injectable and macroporous CPC-RGD constructs, and test these hypotheses: (1) hESC-derived MSCs encapsulated in degradable microbeads and incorporated into CPC will synthesize the most amount of bone matrix, followed by hUCMSCs. The gold-standard hBMSCs will make the least bone matrix; (2) RGD-grafted CPC will greatly enhance cell function and bone matrix synthesis, without compromising the CPC injectability and the mechanical properties, which will match the reported strength of cancellous bone. Aim 2 will investigate the hESC-CPC-RGD constructs for bone regeneration in animal model, and test these hypotheses: (1) New bone volume, mineral density, and blood vessel density generated by CPC-RGD with hESC-derived MSCs will be the highest, followed by hUCMSCs. Both of them will far exceed those generated by hBMSCs; (2) hESC-CPC- RGD will be completely replaced by new bone across the entire critical-sized cranial defect at six months; (3) hESC-CPC-RGD will induce much more new bone than that without RGD. This project will yield ground- breaking knowledge on hESC encapsulation in CPC, hESC interaction with RGD-CPC and the guidance of hESCs for osteodifferentiation, and bone regeneration in animal model comparing hESCs, hUCMSCs, and hBMSCs side-by-side. The new stem cell paste can be used in minimally-invasive surgeries, fill complex- shaped defects, and be easily shaped for esthetics in dental and craniofacial applications. The novel hESC- CPC-RGD construct is expected to have wide orthopedic and craniofacial applications, with greatly enhanced bone regeneration to improve the health and quality of life for millions of people. If indeed, hESCs delivered via CPC are superior in osteogenesis compared to the gold-standard hBMSCs, which is anticipated to be shown by this project for the first time, the results will broadly impact the regenerative medicine field. PUBLIC HEALTH RELEVANCE: Despite the high promise of human embryonic stem cells (hESCs), there has been no report on hESC seeding with injectable calcium phosphate constructs for bone regeneration. The proposed research will investigate hESCs for bone tissue engineering via novel injectable calcium phosphate scaffolds for the first time, and establish ground-breaking knowledge on hESC guidance via peptide-calcium phosphate cement scaffold to enhance hESC function and bone regeneration in animal model. The novel hESC-calcium phosphate construct is expected to have a wide range of craniofacial and orthopedic applications, with greatly enhanced bone regeneration capability to improve the health and quality of life for millions of people.