Objective To investigate the osteogenic ability of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan (DS/rhBMP2/CS) nano microspheres combined with coralline hydroxyapatite (CHA) in repair of segmental bone defects.Methods DS/rhBMP2/CS microspheres prepared by ionic crosslinking method were adsorbed into CHA by lyophilization.Seventy-two New Zealand rabbits were randomly divided into 3 equal groups after they had been made into models of bone defect at the right radius.The defects in the 3 groups were implanted respectively with CHA,rhBMP2/CHA and DS/rhBMP2/ CS/CHA.Another 18 animals served as a blank control group.Blood and bone samples were obtained at 4,8 and 12 weeks after implantation.The serum BGP was detected,and the bone grafts were scanned by micro-CT for calculation of volume ratio of the new bone.Hematoxylin and eosin (HE) staining was performed after bone decalcification.Results All the 72 animals recovered well without any infection or graft exposure.Gross observation at postoperative 12 weeks showed that the DS/rhBMP2/CS/CHA group was the best in the quality,quantity and strength of the new bone,as well as in the healing of bone defects.The serum levels of bone gamma-carboxyglutamic-acid-containing protein at all time points in the DS/rhBMP2/CS/CHA group were significantly higher than those in the CHA and rhBMP2/CHA groups (P ＜ 0.05).Micro-CT scanning demonstrated obvious progress in bone formation,cortical bone and marrow cavity at all time points in the DS/rhBMP2/CS/CHA group which showed significantly faster bone reconstruction synchronized with material degradation and significantly higher volume ratio of the new bone than the other 2 groups (P ＜ 0.05).Histological examinations showed better morphology of mature cortical bone and new marrow cavity at all time points in the DS/rhBMP2/CS/CHA group than in the other 2 groups.Conclusion Since DS/rhBMP2/CS/CHA possesses a better mechanism of sustained-releasing rhBMP2 to induce bone formation because of its reticular and hole-hole-connected structure,it may perform better in repairing segmental bone defects than simple CHA or rhBMP2/CHA.

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Angelica sinensis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Protein Isoforms ; Temperature