Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Chinese Academy of Sciences, Hong Kong; Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong.
Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong; The Sir Yue-Kong Pao Cancer Centre, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong.
Bone. 2024 Jun;183:117094. doi: 10.1016/j.bone.2024.117094. Epub 2024 Apr 4.
The present study aimed to establish and evaluate a preclinical model of steroid-associated osteonecrosis (SAON) in mice. Sixteen 24-week-old male C57BL/6 mice were used to establish SAON by two intraperitoneal injections of lipopolysaccharide (LPS), followed by three subcutaneous injections of methylprednisolone (MPS). Each injection was conducted on working day, with an interval of 24 h. Six cycles of injections were conducted. Additional twelve mice (age- and gender-matched) were used as normal controls. At 2 and 6 weeks after completing induction, bilateral femora and bilateral tibiae were collected for histological examination, micro-CT scanning, and bulk RNA sequencing. All mice were alive until sacrificed at the indicated time points. The typical SAON lesion was identified by histological evaluation at week 2 and week 6 with increased lacunae and TUNEL+ osteocytes. Micro-CT showed significant bone degeneration at week 6 in SAON model. Histology and histomorphometry showed significantly lower Runx2+ area, mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR/BS), type H vessels, Ki67+ (proliferating) cells, and higher marrow fat fraction, osteoclast number and TNFα+ areas in SAON group. Bulk RNA-seq revealed changed canonical signaling pathways regulating cell cycle, angiogenesis, osteogenesis, and osteoclastogenesis in the SAON group. The present study successfully established SAON in mice with a combination treatment of LPS and MPS, which could be considered a reliable and reproducible animal model to study the pathophysiology and molecular mechanism of early-stage SAON and to develop potential therapeutic approaches for the prevention and treatment of SAON.
本研究旨在建立并评估一种小鼠类固醇相关性骨坏死（SAON）的临床前模型。使用 16 只 24 周龄雄性 C57BL/6 小鼠，通过两次腹腔内注射脂多糖（LPS），随后进行三次皮下注射甲泼尼龙（MPS），建立 SAON 模型。每次注射均在工作日进行，间隔 24 小时。共进行 6 个周期的注射。另外 12 只（年龄和性别匹配）小鼠作为正常对照组。在诱导完成后 2 周和 6 周，采集双侧股骨和双侧胫骨进行组织学检查、微 CT 扫描和大量 RNA 测序。所有小鼠均存活至指定时间点处死。在第 2 周和第 6 周的组织学评估中，通过增加的腔隙和 TUNEL+成骨细胞，鉴定出典型的 SAON 病变。微 CT 显示在 SAON 模型中第 6 周时出现明显的骨退变。组织学和组织形态计量学显示，SAON 组的 Runx2+面积、矿化表面（MS/BS）、矿化率（MAR）、骨形成率（BFR/BS）、H 型血管、Ki67+（增殖）细胞显著降低，骨髓脂肪分数、破骨细胞数量和 TNFα+面积显著升高。大量 RNA-seq 显示，在 SAON 组中，调节细胞周期、血管生成、成骨和破骨细胞生成的典型信号通路发生改变。本研究成功建立了 LPS 和 MPS 联合治疗的小鼠 SAON 模型，可作为研究早期 SAON 的病理生理学和分子机制以及开发预防和治疗 SAON 的潜在治疗方法的可靠和可重复的动物模型。
