Objective To explore the biomechanic effects of multi ple freeze-thaw on human allograft tendons. Methods Thirty tendons (24 flexor digitorum superficial is tendons and 6 flexor poll icis longus tendons) were harvested from 3 fresh cadaver donors and were divided into 6 groups randomly (fresh group; 1 cycle, 2 cycle, 3 cycle, 5 cycle, and 10 cycle freeze-thaw groups). There was 4 flexor digitorum superficial is tendons and 1 flexor poll icis longus tendon in each group. The structural and mechanical properties as well as viscoelastic change were estimated. Results The results of the structural and mechanical properties in 1 cycle, 2 cycle, and 3 cycle freeze-thaw groups were similar to that of the fresh group (P gt;0.05). The tendons in 5 cycle and 10 cycle freeze-thaw groups showed a significantly lower ultimate load and maximum stress when compared with those of fresh group (P lt; 0.05), but there was no significant difference in maximum tensile or maximum strain (P gt; 0.05). Moreover, the tendons in 5 cycle and 10 cycle freeze-thaw groups had a significant increase in viscoelastic properties when compared with fresh group (P lt; 0.05). Conclusion In the cryopreservation of tendon allografts, the cycle of freeze-thaw should not exceed 3 times. Multiple cycle freeze-thaw will weaken the biomechanical properties of tendon allografts, which make grafts easier to fatigue or even rupture.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.