[EXPERIMENTAL STUDY ON OSTEOGENESIS OF SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO]

To investigate the osteogenic differentiation potential and the biological features of synovium-derived mesenchymal stem cells (SMSCs) in vitro and to observe the osteogenic capability of the composite scaffolds constructed with SMSCs and hydroxylapatite/chitosan/poly L-latic acid (HA/CS/PLLA) in vivo.

SMSCs were separated and cultured with adherent method and enzymatic digestion method. Specific phenotypes of SMSCs were detected by flow cytometry after purification. Then, SMSCs were identified by oil red O staining, alkaline phosphatase (ALP) staining, and alizarin red staining after adipogenic and osteogenic induction, respectively. In vitro experiments: the expressions of osteogenic related genes [osteocalcin (OCN), collagen type I, ALP, and Runx-2] were detected by real-time fluorescent quantitative PCR at 1, 7, 14, 21, and 28 days after osteogenic induction; ALP activities were also determined by ELISA at 1, 3, 5, 7, 9, and 11 days after osteogenic induction; meanwhile, extracellular matrix calcium mineralization was detected by alizarin red S method at 7, 14, 21, and 28 days after osteogenic induction; the normal SMSCs were harvested as control group. In vivo experiments: Twenty-four Sprague Dawley (SD) rats were randomly divided into experimental group (n = 12) and control group (n = 12). The 3rd passage SMSCs were seeded on HA/CS/PLLA to construct composite scaffolds, after adhesion for 72 hours in vitro, the composite scaffolds were implanted into the right thigh muscle of 12 SD rats as experimental group; HA/CS/PLLA was implanted into the right thigh muscle of the other 12 SD rats as control group. At 4 and 8 weeks after implantation, the scaffolds were harvested for X-ray film and histological examination to observe ectopic bone formation.

The positive rates of CD147, CD90, CD105, and CD44 were more than 95%, while the positive rates of CD117, CD34, CD14, and CD45 were less than 10%. Oil red O staining demonstrated red lipid droplets in the cytoplasm, and alizarin red staining showed flaky red calcifications, and cytoplasm was dyed brown by the ALP staining. The mRNA expressions of collagen type I, ALP, and Runx-2 were significantly increased at 7 days after osteogenic induction, and OCN mRNA expression was significantly increased at 14 days after osteogenic induction; ALP activity was significantly higher at 5, 7, 9, 11 days after osteogenic induction in the SMSC-induced group than control group and reached a maximum at 7 days (P < 0.05). Calcium mineralization was significantly enhanced at 14 days after osteogenic induction, and gradually increased with time (P < 0.05); moreover, it was significantly higher in the SMSC-induced group than control group (P < 0.05). X-ray and histological examination demonstrated that the new bone tissues formed in 2 groups, but bone formation content of the experimental group was significantly more than that of the control group at 4 and 8 weeks after implantation (P < 0.05).

SMSCs can be induced into osteoblasts both in vitro and in vivo, so SMSCs might be a promising seed cells for bone tissue engineering.