Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, QLD 4059, Brisbane, Australia; Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University Munich, Lindwurmstraße 4, 80337 Munich, Germany; Department of Orthopedics for the University of Regensburg, Asklepios Klinikum Bad Abbach, Kaiser-Karl V.-Allee 3, 93077 Bad Abbach, Germany.
Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, QLD 4059, Brisbane, Australia; Orthopedic Center for Musculoskeletal Research, University of Wuerzburg, Koenig-Ludwig-Haus, Brettreichstr. 11, 97074 Wuerzburg, Germany.
Biomaterials. 2018 Jul;171:230-246. doi: 10.1016/j.biomaterials.2018.04.030. Epub 2018 Apr 16.
Existing preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model.
Orthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NOD-scid and NSG mice. NSG mice were then bone marrow transplanted with human CD34 cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA).
After harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples.
OhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies.
现有的临床前鼠模型通常无法预测抗癌药物的效果。为了最大限度地减少鼠宿主与体内异种移植平台人骨肿瘤之间的种间差异，我们组织工程构建了一种新型的原位人源化骨模型。
通过 3D 打印医用聚己内酯支架，构建组织工程化的原位人源化骨构建体（ohTEBC），支架中接种人成骨细胞并嵌入含有人脐静脉内皮细胞（HUVEC）的聚乙二醇水凝胶中。构建体然后植入 NOD-scid 和 NSG 小鼠的股骨中。然后，NSG 小鼠接受人 CD34 细胞的骨髓移植。通过直接注射 Luc-SAOS-2 细胞，在 ohTEBC 中诱导人骨肉瘤（OS）生长。采集组织进行骨基质和骨髓形态分析以及肿瘤生物学研究。利用组织微阵列（TMA）分析人源化 OS 中的肿瘤标志物表达，并与 68 名 OS 患者的表达进行相关性分析。
取出股骨后，微计算机断层扫描和免疫组织化学染色显示了一个具有所有人类骨骼特征的器官。在原来的小鼠股骨周围形成了新的骨小梁，周围有骨皮质。人特异性（hs）I 型胶原（hs Col-I）染色显示了人细胞外骨基质的产生。细胞核人核有丝分裂装置蛋白 1（hs NuMa）阳性染色证明了骨基质中存在的骨细胞来源于人类。流式细胞术证实了人造血细胞的存在。注射 Luc-SAOS-2 细胞后，原发性肿瘤和肺转移发展。安乐死后的组织学分析显示出成骨性 OS 的典型特征。此外，肿瘤利用先前植入的 HUVEC 进行血管生成。肿瘤标志物的表达与人类患者相似。此外，最近发现的肌肉骨骼基因 C12orf29 在最常见的 OS 患者样本亚型中表达。
ohTEBC 代表了人源化 OS 生长的合适原位微环境，为股骨是最常见的 OS 部位提供了新的转化方向。新开发和验证的临床前模型允许对原发性骨肿瘤和其他骨恶性肿瘤进行可控和预测性标志物研究。
