title: Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo.
abstract: 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.
authors.affiliation: East China University of Science and Technology, Shanghai, PR China.
authors.forename: Huanjun
authors.initials: H
authors.lastname: Zhou
authors.forename: Jiangchao
authors.initials: J
authors.lastname: Qian
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Wantong
authors.initials: W
authors.lastname: Yao
authors.forename: Changsheng
authors.initials: C
authors.lastname: Liu
authors.forename: Jianguo
authors.initials: J
authors.lastname: Chen
authors.forename: Xuehua
authors.initials: X
authors.lastname: Cao
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D048271:Chitosan / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017550:Spectroscopy, Fourier Transform Infrared; D013455:Sulfur / Q000378:metabolism; D013552:Swine
chemical_list: C539268:2-N, 6-O-sulfated chitosan; D055396:Bone Morphogenetic Protein 2; D013455:Sulfur; D048271:Chitosan; D000469:Alkaline Phosphatase