Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America.
Department of Molecular and Cellular Medicine, Texas A&M College of Medicine, Texas A&M University, College Station, Texas, United States of America.
PLoS One. 2022 Jun 9;17(6):e0269571. doi: 10.1371/journal.pone.0269571. eCollection 2022.
The dog is an underrepresented large animal translational model for orthopedic cell-based tissue engineering. While chondrogenic differentiation of canine multipotent stromal cells (cMSCs) has been reported using the classic micromass technique, cMSCs respond inconsistently to this method. The objectives of this study were to develop a three-dimensional (3D), serum-free, Collagen Type I system to facilitate cMSC chondrogenesis and, once established, to determine the effect of chondrogenic growth factors on cMSC chondrogenesis. Canine MSCs were polymerized in 100 μL Collagen Type I gels (5 mg/mL) at 1 x 106 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology in 10 various chondrogenic media. Four media were selected for additional in-depth analyses via lactate dehydrogenase release, total glycosaminoglycan content, qPCR (COL1A1, COL2A, SOX9, ACAN, BGLAP and SP7), immunofluorescence, and TUNEL staining. In the presence of dexamethasone and transforming growth factor-β3 (TGF-β3), both bone morphogenic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) generated larger chondrogenic constructs, although BMP-2 was required to achieve histologic characteristics of chondrocytes. Chondrogenic medium containing dexamethasone, TGF-β3, BMP-2 and bFGF led to a significant decrease in lactate dehydrogenase release at day 3 and glycosaminoglycan content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were induced in response to dexamethasone, TGF-β3, BMP-2 and bFGF. Collagen Type II and X were detected in all groups via immunofluorescence. Finally, TUNEL staining was positive in constructs lacking BMP-2 or bFGF. In conclusion, the 3D, serum-free, Collagen Type-I assay described herein proved useful in assessing cMSC differentiation and will serve as a productive system to characterize cMSCs or to fabricate tissue engineering constructs for clinical use.
犬是一种在矫形细胞基组织工程中代表性不足的大型动物转化模型。虽然犬多能基质细胞（cMSCs）的软骨分化已通过经典的微团技术进行了报道，但 cMSCs 对该方法的反应不一致。本研究的目的是开发一种三维（3D）、无血清的 I 型胶原系统，以促进 cMSC 软骨形成，一旦建立，确定软骨形成生长因子对 cMSC 软骨形成的影响。犬 MSCs 在 100μL I 型胶原凝胶（5mg/mL）中以 1×106 细胞/构建物的浓度聚合。通过形态计量学、活/死染色和组织学评估构建物，在 10 种不同的软骨形成培养基中。选择了四种培养基用于通过乳酸脱氢酶释放、总糖胺聚糖含量、qPCR（COL1A1、COL2A、SOX9、ACAN、BGLAP 和 SP7）、免疫荧光和 TUNEL 染色进行进一步深入分析。在存在地塞米松和转化生长因子-β3（TGF-β3）的情况下，骨形态发生蛋白-2（BMP-2）和碱性成纤维细胞生长因子（bFGF）均生成更大的软骨形成构建物，尽管 BMP-2 是实现软骨细胞组织学特征所必需的。含有地塞米松、TGF-β3、BMP-2 和 bFGF 的软骨形成培养基可显著降低第 3 天的乳酸脱氢酶释放，并且这些构建物中的糖胺聚糖含量在第 3、10 和 21 天显著增加。地塞米松、TGF-β3、BMP-2 和 bFGF 均诱导成骨和软骨转录物。通过免疫荧光在所有组中均检测到 II 型和 X 型胶原蛋白。最后，在缺乏 BMP-2 或 bFGF 的构建物中 TUNEL 染色呈阳性。总之，本文所述的 3D、无血清的 I 型胶原测定法在评估 cMSC 分化方面非常有用，并且将作为一种有价值的系统来表征 cMSCs 或制造用于临床应用的组织工程构建物。
