| Bone morphogenetic protein2(BMP-2) is one member of the Bonemorphogenetic proteins, which also belongs to transforming growth factor beta(TGF-β) superfamily. BMP-2plays an important role in the stimulating osteoblastdifferentiation and the development of bone. BMP-2can only last its bioactivitiesabout several seconds or several minutes in vivo. And a idea carrier can maintain itsbioactivities and achieve to sustain release, however there always is low affintybetween carrier and BMP-2, which causes quick dumping. In recent years, reseachershave made a lot effort on modification of carriers or BMP-2to improve the affintybetween them. On the other hand, silk fibroin(SF) has been widely used for theresearch of bone tissue engineering and drug delivery system because of its preferablymechanical properties and biocompatibility.In this study, BMP-2were expressed in the E. coli strain BL21which is aprokaryotic expression system, then the proteins were purified and refolded, and alsotest its bioactivity. The SF scaffolds were prepared by freeze-drying, and two specificbinding BMP-2silk fibroin scaffords were prepared by couping heparin with twodifferent methods, one heparinized silk scaffold named HP/SF was directly coupledheparin by carbodiimide method, and another heparinized silk scaffold namedHP/L-SF first modified with lysine was also coupling heparin with the same way.Properties of these scaffolds were investigated by SEM, FTIR, water absorption andporosity, etc. And the amounts of heparin, capibility of adsorption and release BMP-2were also detected. Besides, we test the cytotoxicity of two specific binding BMP-2silk fibroin scaffords and their biocompatibility when conbined with BMP-2.Molecular weight of BMP-2was about20kDa as expected, BMP-2shows highbiological activity for cell differentiation with the low concentration of BMP-2. Theresults of SEM and porosity showed that silk fibroin scaffords had high porosity,interconnected pores with pore diameter which range from100to200μm. FTIRresults showed that SF was tansformed into β-sheet conformation. The amount ofincorporated heparin were1.80±0.14μg/mg and1.10±0.22μg/mg respectively. After BMP-2absorption, results showed that the two specific binding BMP-2silkfibroin scaffords could bind BMP-2well, compared with BMP-2absorption capacityof SF which is12.21±0.52μg, HP/SF and HP/L-SF have stronger BMP-2absorptioncapacity, the amount of BMP-2absorption is15.15±0.93μg,19.65±1.81μgrespectively. HP/SF and HP/L-SF also achieve to maintain more sustained way torelease. Compare with dumping release rate(about50.41%) on the first day, HP/SFand HP/L-SF both bave much slower release rate (29.90%,26.82%), after7days’release, about78.41%BMP-2have released from SF, but HP/SF and HP/L-SF havereleased53.53%and40.97%respectively.The Results of biocompatibility shows thattwo specific binding BMP-2silk fibroin scaffords could improve the MG-63celldifferentiation with higher ALP activity than silk fibroin scaffords after loadingBMP-2，and BMP-2loaded HP/L-SF shows higher ALP activity when compared withBMP-2loaded HP/SF.Aiming at improving the affinty between BMP-2and its carrier, this studyprepare two specific binding BMP-2silk fibroin scaffords by heparinization, theresults show that the two specific binding BMP-2silk fibroin scaffords possessedexcellent biocompatibility, and both were preferably BMP-2carriers. This study mayprovide an interesting idea in the fabrication of other BMP-2carriers. |
