Ueno Masaya, Zhang Ning, Hirata Hirohito, Barati Danial, Utsunomiya Takeshi, Shen Huaishuang, Lin Tzuhua, Maruyama Masahiro, Huang Ejun, Yao Zhenyu, Wu Joy Y, Zwingenberger Stefan, Yang Fan, Goodman Stuart B
Department of Orthopaedic Surgery, Stanford University, Stanford, CA, United States.
Department of Orthopaedic Surgery, Faculty of Medicine, Saga University, Saga, Japan.
Front Bioeng Biotechnol. 2021 Oct 19;9:755964. doi: 10.3389/fbioe.2021.755964. eCollection 2021.
Mesenchymal stem cell (MSC)-based therapy and novel biomaterials are promising strategies for healing of long bone critical size defects. Interleukin-4 (IL-4) over-expressing MSCs within a gelatin microribbon (µRB) scaffold was previously shown to enhance the bridging of bone within a critical size femoral bone defect in male Balb/c mice. Whether sex differences affect the healing of this bone defect in conjunction with different treatments is unknown. In this study, we generated 2-mm critical-sized femoral diaphyseal bone defects in 10-12-week-old female and male Balb/c mice. Scaffolds without cells and with unmodified MSCs were implanted immediately after the primary surgery that created the bone defect; scaffolds with IL-4 over-expressing MSCs were implanted 3 days after the primary surgery, to avoid the adverse effects of IL-4 on the initial inflammatory phase of fracture healing. Mice were euthanized 6 weeks after the primary surgery and femurs were collected. MicroCT (µCT), histochemical and immunohistochemical analyses were subsequently performed of the defect site. µRB scaffolds with IL-4 over-expressing MSCs enhanced bone healing in both female and male mice. Male mice showed higher measures of bone bridging and increased alkaline phosphatase (ALP) positive areas, total macrophages and M2 macrophages compared with female mice after receiving scaffolds with IL-4 over-expressing MSCs. Female mice showed higher Tartrate-Resistant Acid Phosphatase (TRAP) positive osteoclast numbers compared with male mice. These results demonstrated that sex differences should be considered during the application of MSC-based studies of bone healing.
基于间充质干细胞（MSC）的治疗和新型生物材料是治疗长骨临界尺寸骨缺损的有前景的策略。先前研究表明，在明胶微带（µRB）支架内过表达白细胞介素-4（IL-4）的间充质干细胞可增强雄性Balb/c小鼠临界尺寸股骨骨缺损处的骨桥接。尚不清楚性别差异是否会与不同治疗方法共同影响这种骨缺损的愈合。在本研究中，我们在10至12周龄的雌性和雄性Balb/c小鼠中制造了2毫米临界尺寸的股骨干骺端骨缺损。在造成骨缺损的初次手术后立即植入无细胞和未修饰间充质干细胞的支架；植入过表达IL-4的间充质干细胞的支架在初次手术后3天植入，以避免IL-4对骨折愈合初始炎症期的不利影响。初次手术后6周对小鼠实施安乐死并收集股骨。随后对缺损部位进行显微计算机断层扫描（µCT）、组织化学和免疫组织化学分析。植入过表达IL-4的间充质干细胞的µRB支架在雌性和雄性小鼠中均增强了骨愈合。与接受过表达IL-4的间充质干细胞支架的雌性小鼠相比，雄性小鼠显示出更高的骨桥接程度以及碱性磷酸酶（ALP）阳性面积、总巨噬细胞和M2巨噬细胞数量增加。与雄性小鼠相比，雌性小鼠显示出更高的抗酒石酸酸性磷酸酶（TRAP）阳性破骨细胞数量。这些结果表明，在基于间充质干细胞的骨愈合研究应用中应考虑性别差异。
