Musumeci Giuseppe, Mobasheri Ali, Trovato Francesca Maria, Szychlinska Marta Anna, Graziano Adriana Carol Eleonora, Lo Furno Debora, Avola Rosanna, Mangano Sebastiano, Giuffrida Rosario, Cardile Venera
Department of Bio-Medical Sciences, Human Anatomy and Histology Section, School of Medicine, University of Catania, Catania, Italy.
Department of Veterinary Preclinical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom; Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, Arthritis Research UK Pain Centre, Medical Research Council and Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom; Center of Excellence in Genomic Medicine Research (CEGMR), King Fahd Medical Research Center (KFMRC), King AbdulAziz University, Jeddah, Kingdom of Saudi Arabia.
Acta Histochem. 2014 Oct;116(8):1407-17. doi: 10.1016/j.acthis.2014.09.008. Epub 2014 Oct 11.
The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called 'cell pellets'. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.
本研究的首要目的是通过自组装过程确定脂肪组织来源的间充质干细胞（MSCs）向软骨细胞分化的最合适时间。为此，利用免疫组织化学和蛋白质印迹分析，在MSCs软骨分化的不同时间点（7、14、21和28天）研究了一些软骨细胞标志物的表达，如I型胶原、II型胶原、RUNX2和润滑素。本研究的第二个目的是证明润滑素的表达，如II型胶原的表达，可能是检测天然自组装构建体（称为“细胞球”）中软骨细胞健康状况和活力的生物标志物。采用组织学（苏木精和伊红染色）和组织化学（阿尔辛蓝染色）方法评估MSCs的软骨分化。结果显示，与未用软骨生成培养基培养的MSCs（CD44、CD90和CD105阳性；CD45、CD14和CD34阴性）相比，21天后分化的软骨细胞能够产生大量的I型胶原、II型胶原和润滑素，提示透明软骨形成。在分化阶段，细胞显示成骨细胞表达的RUNX2蛋白表达降低。我们的研究表明，21天是植入由脂肪组织来源的MSCs分化而来的软骨细胞的最佳时间。这一信息可能有助于未来基于细胞的关节软骨退行性疾病修复治疗的发展。
