Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro-shi, Hokkaido 080-8555, Japan.
Department of Clinical Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi city, Yamaguchi 753-8515, Japan.
Res Vet Sci. 2019 Aug;125:345-350. doi: 10.1016/j.rvsc.2019.07.005. Epub 2019 Jul 10.
In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 × 10, 2 × 10, and 5 × 10 equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell substrata for 4 days to allow formation of clusters. The medium was replaced by chondrogenic culture medium. After chondrogenic culture for 7, 14 and 21 days, analysis of collagen type II alpha 1 gene (COL2A1) gene expression and observation of chondrogenic aggregates by scanning electron microscopy (SEM) were performed. SEM showed size-controlled cell clusters and increasing extracellular matrix over time when using 5 × 10 cells. The expression of COL2A1 on day 7 and 14 with 5 × 10 cells was significantly higher than that of conventional pellet culture with 2 × 10 cells. Histological evaluation by immunohistochemical staining for type II collagen (ColII) was performed after chondrogenic culture for 7 days. The clusters showed wide distribution of ColII. The results suggest that the zirconia substrata have the potential to enhance the chondrogenic differentiation of equine BMMSCs, allowing effective equine cartilage tissue engineering without xenobiotic materials.
在人类软骨组织工程中，三维氧化锆基底具有产生许多大小均匀的细胞簇的潜在优势，且无需使用异源材料，从而便于临床应用。本研究旨在评估氧化锆多孔三维微井基底在体外诱导马骨髓间充质干细胞（BMMSCs）向软骨分化的可能性。在常规培养基中，将来自五匹纯血马的 8×10、2×10 和 5×10 个马 BMMSCs 分别接种在氧化锆微井基底上，培养 4 天以形成细胞簇。然后将培养基更换为软骨形成培养基。在软骨形成培养 7、14 和 21 天后，通过扫描电子显微镜（SEM）分析Ⅱ型胶原α 1 基因（COL2A1）的基因表达，并观察软骨聚集物。SEM 显示，当使用 5×10 个细胞时，可实现大小可控的细胞簇形成，并随着时间的推移增加细胞外基质。与使用 2×10 个细胞的常规微球培养相比，使用 5×10 个细胞在第 7 和 14 天 COL2A1 的表达显著更高。在软骨形成培养 7 天后，通过Ⅱ型胶原蛋白（ColII）的免疫组织化学染色进行组织学评价。这些细胞簇显示出 ColII 的广泛分布。结果表明，氧化锆基底具有增强马 BMMSCs 软骨分化的潜力，可在无需使用异源材料的情况下有效进行马软骨组织工程。
