Subcutaneous transplantation of marrow-derived murine mesenchymal stem cells cultivated in alginate and their chondrogenesis

Mesenchymal stem cells are appropriate candidates to treat diseases including articular cartilage defects. There are plenty of researches being conducted to make the application of these cells possible. The purpose of this study was to cultivate murine mesenchymal stem cells [MSCs] in alginate gel and transplant them subcutaneously to immuno-suppressed rats to examine their chondrogenic potential in vivo. 4-6 week old NMRI mice were sacrificed and their bone marrow cells were cultivated in 6-cell plates at the density of 500 cell/cm[2]. The pure fibroblastic cells appeared after two passages. 2X10[6] flbroblastic cells were mixed with 1 ml of alginate solution and converted into gel by being exposed to calcium chloride solution. MSCs-embedded alginate gel were then transplanted subcutaneously to 6 rats that had received an immunosuppressive drug [cyclosporine] for transplant rejection to be avoided. 5 weeks after transplantation, the alginate gels were removed and evaluated by histochemistry, RT-PCR for certain cartilage markers, and transmission electron microscopy. 5 weeks after transplantation, the skin was incised and the alginate gel with its surrounding vascular connective tissue were removed. Tuloidine blue staining indicates that the cells within the gel assumed oval morphology and occupied lacuna-like cavities. RT-PCR analysis revealed that in these cells the mRNA of some cartilage markers such as collagen II [the marker of hyaline cartilage], collagen X [hypertrophied chondrocyte marker in osteogenesis], and aggreacan were largely produced. Ultra-thin sections analysis showed that the cells within the lacuna-like cavity of alginate gel contain a large amount of expanded rough endoplasmic reticulum and secret fibrillar extra cellular matrix. Transplanted murine MSCs cultivated in alginate gel can differentiate into hyaline cartilage with the sign of osteogenic initiation

[ comparative study between the structure of cartilage tissue produced from murine MSC differentiation and hyaline costal cartilage]

Vitro cartilage differentiation of mesenchymal stem cells [MSCs] has been noticed in several investigations. In this regard, almost always molecular differentiation of the cells has been examined, while structural and morphological differentiation of them has been ignored. Therefore, the present study examines the structure and ultrastructure of the cartilage differentiated from murine MSCs compared with that of costal cartilage. 2x 10[5] MSCs isolated from the bone marrow of NMRI mice were pleted by centrifugation and cultured for 21 days in chondrogenic medium. At the end of cultivation period, occurrence of chondrogenic differentiation was confirmed by reverse transcriptase-polymerase chain reaction [RT-PCR] analysis for some cartilage-specific genes. To compare the structure of differentiated tissue with that of natural cartilage, the cartilage was differentiated from MSCs and the cartilage obtained from the same murine rib was prepared for transmission electron microscopy [TEM]. Structural studies indicated that similar to the costal cartilage, the cartilage produced from differentiation of perichondrium-like layer was formed. According to the microscopic images, in contrast to costal chondrocytes, the differentiated cells had euchromatic nucleus and their cytoplasm contained plenty of the organelles involved in active cell secretion. Furthermore, intercellular matrix in differentiated cartilage had a fibrillar appearance. Our results indicated that the structure of cartilage produced in micro mass culture system is somewhat different from that of costal cartilage. The cells from differentiated tissue seemed to be more active than those from costal cartilage