Human Embryonic Stem Cells in Calcium Phosphate Constructs for Bone Regeneration

描述(申请人提供)：美国每年有700万人骨折。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加。人类胚胎干细胞(HESCs)提供无限的干细胞供应，具有很高的骨再生潜力。然而，目前还没有通过注射磷酸钙将人胚胎干细胞用于骨组织工程的报道。虽然人骨髓间充质干细胞(HBMSCs)是有用的，但它们的获取需要侵入性的过程，而且由于年龄和疾病的原因，它们的增殖和分化潜力很大。因此，本项目的目标是：(1)研究可注射磷酸钙骨水泥(CPC)中的人胚胎干细胞(HESCs)用于骨工程，并与人脐带间充质干细胞(HUCMSCs)和人骨髓间充质干细胞(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hESCs的增殖和成骨分化；(3)设计一种用于hESC包埋的RGD-CPC支架，以促进骨再生。虽然精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp，RGD)已被用于其他支架，但其在CPC中的应用尚未见报道。目的1体外研究hESC在可注射和大孔CPC-RGD支架中的包埋和分化，并验证以下假设：(1)hESC来源的MSCs被包裹在可降解的微球中并掺入CPC中将合成最多的骨基质，其次是hUCMSCs。金标的hBMSCs将生成最少的骨基质；(2)RGD-CPC可显著增强细胞功能和骨基质的合成，而不影响CPC的可注射性和力学性能，与文献报道的松质骨强度相当。目的研究hESC-CPC-RGD构建的骨再生动物模型，并验证以下假设：(1)hESC来源的MSCs构建的CPC-RGD所生成的新生骨体积、骨密度和血管密度最高，其次是hUCMSCs。(2)hESC-CPC-RGD在6个月后将完全被新骨替代；(3)hESC-CPC-RGD在整个临界大小的颅骨缺损中诱导出的新骨明显多于不含RGD的颅骨。该项目将在hESC在CPC中的包裹、hESC与RGD-CPC的相互作用以及hESCs对成骨分化的指导方面产生开创性的知识，以及在动物模型中比较hESCs、hUCMSCs和hBMSCs的骨再生。这种新的干细胞糊剂可用于微创手术，填补复杂形状的缺陷，并可轻松成形用于牙科和颅面应用的美学。这种新型的hESC-CPC-RGD结构有望在骨科和颅面部得到广泛的应用，极大地增强骨再生，改善数百万人的健康和生活质量。如果真的是这样，通过CPC输送的hESCs在成骨方面比黄金标准的hBMSCs更好，预计这将是本项目首次展示，结果将广泛影响再生医学。 菲尔德。

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.