| Bone morphogenetic protein-2(BMP-2)is a potent protein drug for bone repairing.However,BMP-2 is easily degraded and inactivated in vivo.The use of BMP-2 always accompanies with high risk of side-effects and high price,which limits the clinic application.Heparan sulfate(HS)can regulate the storage,activation and release of proteins in human body through the sulfated glycosylation on the molecular chain.However,the extremely complex structure of HS limits its clinical application.Hererin,we are inspired by the HS regulates protein activity through its diverse structures.Based on our previous work,a series of glycopolymers with different structures were designed and prepared to mimetic HS’s structure and function.The interaction between glycopolymers and BMP-2,as well as the regulation of osteogenic differentiation were studied.(1)An azido glycosyl group was synthesized from N-acetyl-D-glucosamine.Then,methyl methacrylate and hydroxyethyl methacrylate as the hydrophobic and the hydrophilic units were used to obtain block-and random-type main chains by reversible addition-fragmentation chain transfer polymerization.After glycosylation and sulfated modification,a series of 6-O and3,4,6-O sulfated glycopolymers were obtained.Their chemical structures were characterized by hydrogen nuclear magnetic resonance spectroscopy,infrared spectroscopy and gel permeation chromatography,respectively.The results show that the molecular weight of the glycopolymers is controllable,ranging from 14 to 16 kDa.Glycopolymers can self-assemble in aqueous solution to form homogenous micelles with the particle size at 50-100 nm.(2)BMP-2 was used as the model protein to evaluate the glycopolymer-protein interactions by a quartz crystal microbalance.The results showed that the unsulfated glycopolymer cannot bind to BMP-2,the 6-O sulfated ones and 3,4,6-O sulfated ones showed strong binding ability with BMP-2.The binding force increased with sulfation degree and amount of sulfated glycosyl groups.The results of circular dichroism showed that the addition of glycopolymer would affect the BMP-2 conformation.This illustrated as the α-helix increased from 17.0% to 57.2%,the β-sheet decreased from 27.7% to 3.2%,the random coil reduced from 41% to 21.3% and the β-turn maintained.These conformational changes may be key factors affecting the activity of BMP-2 protein.(3)The cytotoxicity of glycopolymers was evaluated by MTT assay and live-dead staining experiments.The results showed that the glycopolymers had good cytocompatibility and could promote cell growth.The glycopolymer/BMP-2 complexes can stimulate the expression of osteogenesis-related factors,which shows as up-regulation of alkaline phosphatase expression,increase of cell mineralization and collagen secretion.The osteogenic differentiation is closely related to the molecular structure of glycopolymers.Among them,the 3,4,6-O sulfated one show the most effectively improvements.The dosage of BMP-2 could decrease to 200 ng/mL as compared with the clinical dose(10 μg/mL).In conclusion,the glycopolymers synthesized in this thesis can regulate the activity of BMP-2 and promote osteoblast differentiation.This provides a new path of BMP-2 formulation and clinical application in bone repairing. |
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