Hamamoto Shuichi, Chijimatsu Ryota, Shimomura Kazunori, Kobayashi Masato, Jacob George, Yano Fumiko, Saito Taku, Chung Ung-Il, Tanaka Sakae, Nakamura Norimasa
Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Japan.
Bone and Cartilage Regenerative Medicine, The University of Tokyo, Tokyo, Japan.
J Exp Orthop. 2020 Mar 7;7(1):10. doi: 10.1186/s40634-020-00228-8.
Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946.
Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks.
Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed.
Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.
基于软骨细胞的组织工程一直是治疗软骨损伤的一个有前景的选择。在先前的文献中，TD198946已被证明可促进软骨形成分化，这可能对软骨再生治疗有用。我们的研究旨在探讨TD198946在用TD198946处理的去分化软骨细胞接种的胶原支架中生成工程化软骨的作用。
从迷你猪膝关节分离关节软骨细胞，在二维细胞培养中扩增，随后用于实验。然后将三维微球培养两周。细胞也在I型胶原支架中培养四周。标本用TD198946、骨形态发生蛋白-2（BMP-2）或两者联合培养。通过RUNX1、SOX9、ACAN、COL1A1、COL2A1和COL10A1的基因表达水平、糖胺聚糖含量以及组织学和免疫组织化学特征来确定结果。此外，通过在裸鼠皮下植入四周来评估培养两周的工程化软骨的成熟度。
添加TD198946显示除了ACAN外，在微球和三维支架培养中基因表达水平上调，II型胶原和糖胺聚糖合成增加。TD198946和BMP-2联合培养显示出比单独使用TD198946或BMP-2更高的软骨形成分化。工程化软骨在植入后四周保持其细胞外基质。相比之下，单独用TD198946或BMP-2处理的工程化软骨大多被吸收。
我们的结果表明，TD198946可通过植入前对去分化软骨细胞进行再分化以及促进胶原和糖胺聚糖合成来提高工程化软骨的质量。
