Assessment of Biological Activity of 28-Homobrassinolide via a Multi-Level Comparative Analysis.

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24R-methyl-epibrassinolide (24-EBL) and 24S-ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22R,23R)-28-HBL and inactive (22S,23S)-28HBL, demands a standardized assay system capable of analyzing different synthetic "28-HBL" products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana. These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure-activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

Fabrication and clinical application of easy-to-operate pre-cured CPC/rhBMP-2 micro-scaffolds for bone regeneration.

Bone tissue engineering scaffolds loading growth factors have been considered as the most perspective among all bone substitutes, yet little progress of its clinical translation has been made. The concept of "micro-scaffolds" was proposed in this study to provide a trajectory to the clinical translation of porous scaffolds. Combining CPC and rhBMP-2, a pre-cured CPC/rhBMP-2 micro-scaffold has been successfully developed and further applied as an easy-to-operate filler for bone regeneration in a pilot clinical study. The results demonstrated superior overall performances of CPC/rhBMP-2 microffolds to traditional therapies, with not only shortened repairing time and improved repairing qualities, but also the potential in treating fractures that are most challenging for current therapies. This pilot clinical study of CPC/rhBMP-2 microffolds further promoted the clinical translation of porous scaffolds for bone regeneration, and provided new insights for future development of artificial bone substitutes.

The impact of two different thaw protocols on outcomes of vitrified cleavage-stage embryos transfer.

The objective of this study was to evaluate the gestational results obtained with vitrified-thawed human cleavage-stage embryo by two different thaw protocols. Embryo development was observed to cleavage-stage and embryos were cryopreserved by vitrification on day 3 after oocyte retrieval. 51 cycles were thawed using vitrification warming kit with decreasing concentrations of sucrose in 3 dilutions ( 1.0, 0.5 and 0 mol per L respectively) as group 1, 56 cycles were thawed with decreasing concentrations of sucrose in 5 dilutions ( 0.8, 0.6, 0.33, 0.2 and 0 mol per L respectively) as group 2. Embryo survival (> 50 percent intact blastomeres), complete embryo survival (100 percent intact blastomeres), pregnancy and implantation rates were compared, and development rates the day after thawing were also compared. Multivariate analysis showed a significant difference in embryo immediate morphological survival rate, complete survival and clinical pregnancies rate between the two groups respectively (87.0 vs. 98.6 percent, p=0.000; 71.0 vs 82.0 percent embryo subsequent development rates, mean number of transferred embryos was similar between the two groups. (61.4 vs. 61.3 percent, p=0.502; 2.2 +/ 0.5 vs. 2.4 +/- 0.6, p=0.113). In addition, no differences in implantation rate were observed between two groups (17.7 vs. 25.6 percent, P=0.138). No difference in the multiple pregnancy rates was found among the two groups also.

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo.

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.

[Experimental studies on the porous calcium phosphate cement combined with recombinant human bone morphogenetic protein 2 for bone defects repair].

OBJECTIVE: To study in vitro sustained release behaviour of the recombinant human bone morphogenetic protein 2 (rhBMP-2) from the sample which porous calcium phosphate cement (PCPC) was combined with rhBMP-2, and to evaluate the effect of PCPC/rhBMP-2 composite on repairing bone defect in the animal study. METHODS: rhBMP-2 was absorbed into PCPC by vacuum-adsorption and freeze-dried at -40 degrees C, the PCPC/rhBMP-2 enwrapped with chitosan as the experimental group, the pure PCPC/rhBMP-2 as the control group, then the sustained release of rhBMP-2 from PCPC was determined in simulated body fluid (SBF) by UV-VIS spectrophotometer. At same time, the PCPC/rhBMP-2 composites with chitosan were implanted into the (4.2 mm x 5.0 mm femora defects of rabbits, which were considered as the experimental group, whereas in the control group only PCPC was implanted. The effect of repairing bone defect was evaluated in the 4th and 8th week postoperatively by radiograph and histomorphology. RESULTS: The PCPC have a high absorption efficiency to rhBMP-2, and the release of rhBMP-2 was sustained release system. The release of rhBMP-2 from PCPC in the experimental group (99% after 350 hours) was slower than that in the control group (100% after 150 hours). In the experimental group, the radiological and histomorphological evaluations showed that the interfaces between the materials and host bones became blurred both at 4th and 8th week. The implanted materials were partially absorbed, and the implanted areas exhibited the formation of new bone. In the control group, a little amount of new bones was observed. CONCLUSION: The PCPC shows great clinical potential as a carrier for rhBMP-2. The PCPC/rhBMP-2 composite possesses much potentialities of osteoinductivity and the ability of repairing bone defect, so it can be used as a novel bone substitute clinically.