1 Department of Orthopedics and Trauma Surgery, Medical University of Graz, Graz, Austria.
2 Research Unit for Experimental Neurotraumatology, Department of Neurosurgery, Medical University of Graz, Graz, Austria.
Tissue Eng Part C Methods. 2019 Apr;25(4):197-212. doi: 10.1089/ten.TEC.2018.0368.
Translational studies to elucidate the response of immature bone to biologic and physical stimuli have been held back by the lack of a viable long-term functional bone explant model. This study attempts to bridge this gap between cell culture and animal model studies. In this study, we describe a methodology to derive a 300 μm organotypic femur slice comprising physiological zones (epiphysis and meta-diaphysis) essential for endochondral bone development. The unique capability of slice culture model incorporating enhanced nutrient access to distinct bone tissue components associated with linear bone growth facilitates the investigation of the orchestrated cellular transition of chondrogenic and osteogenic cells involved in endochondral bone development in an setup. Bone slices of 300 μm were prepared from 4-day-old postnatal rats and were viable in culture up to 21 days. On days 7 and 15, an increase in chondrogenic and osteogenic modulations was confirmed in epiphysis, metaphysis, and diaphysis. An increase in osteocytes, osteoblasts, and hypertrophic cells were found at these time points, as well as a noticeable increased expression of chondrogenic and osteogenic markers (collagen II, Runx2, and osteocalcin) confirmed endochondral progression. Osteoclast-mediated bone resorption was demonstrated on day 15 by tartrate-resistant acid phosphatase staining. Attenuated total reflection infrared spectroscopic analyses, furthermore, confirmed a time-dependent increase in phosphate levels, bone minerals, and hydroxyapatite for 15 days. Our establishment of a bone slice culture model closely mimicking the cellular transitions and endochondral microenvironment of a mineralizing bone provides a vital new tool for the elucidation of cellular and endochondral mechanisms of bone development, maturation, and growth plate modulations. The presented model has the potential to be utilized in implementation of preclinical, toxicological, and therapeutic investigations.
将阐明未成熟骨对生物和物理刺激的反应的转化研究受到缺乏可行的长期功能性骨外植体模型的阻碍。本研究试图弥合细胞培养和动物模型研究之间的差距。在这项研究中，我们描述了一种从 300μm 器官型股骨切片中获取的方法，该切片包括生理区（骨骺和骨干），对于软骨内骨发育至关重要。切片培养模型的独特能力结合了增强的营养物质对与线性骨生长相关的不同骨组织成分的获取，促进了在体外条件下参与软骨内骨发育的成软骨和成骨细胞的协调细胞转变的研究。从 4 天大的新生大鼠中制备 300μm 的骨切片，并在培养中保持活力长达 21 天。在第 7 天和第 15 天，在骨骺、骨干和骨干中证实了成软骨和成骨调节的增加。在这些时间点发现了成骨细胞、成骨细胞和肥大细胞的增加，以及软骨内进展的明显增加的成软骨和成骨标志物（胶原 II、Runx2 和骨钙素）的表达。在第 15 天，通过抗酒石酸酸性磷酸酶染色证明了破骨细胞介导的骨吸收。衰减全反射红外光谱分析进一步证实，在 15 天内磷酸盐水平、骨矿物质和羟磷灰石呈时间依赖性增加。我们建立的骨切片培养模型非常类似于矿化骨的细胞转变和软骨内微环境，为阐明骨发育、成熟和生长板调节的细胞和软骨内机制提供了一个重要的新工具。所提出的模型有可能用于实施临床前、毒理学和治疗学研究。
