PubMed · 16122480
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Akira Iyoda. 2005. Invited commentary.. https://doi.org/10.1016/j.athoracsur.2005.05.048
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OBJECTIVE: The aim of this study was to fabricate a novel composite porous scaffold by blending chitosan and gamma-poly(glutamic acid) (gamma-PGA) for the sustained delivery of rhBMP-2. METHODS: Chitosan and gamma-PGA were blended to fabricate a novel porous scaffold by the freeze-gelation method. For comparison, scaffolds made of freeze-dried chitosan, freeze-dried PLLA, and freeze-gelled chitosan were also prepared. The scaffolds were loaded with rhBMP-2, and then the controlled release of rhBMP-2 from the scaffolds was assessed by ELISA. RESULTS: The freeze-gelled chitosan/gamma-PGA scaffold (M0=318.29 ng, k=0.32 d(-1)) gave the most satisfactory release curve, followed by the freeze-gelled chitosan (M0=392.76 ng, k=0.59 d(-1)), freeze-dried chitosan (M0=229.21 ng, k=2.28 d(-1)), and freeze-dried PLLA (M0=8.4 ng, k=482.54 d(-1)) scaffolds. In the stability test, p-dioxane (the solvent for PLLA) seriously deteriorated rhBMP-2, whereas acetic acid (the solvent for chitosan) did not. SIGNIFICANCE: A novel chitosan/gamma-PGA composite scaffold for the controlled release of rhBMP-2 was established, with an enhanced release amount and sustained release behavior. This scaffold has many potential applications in bone regenerative therapies.
Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.
The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. By means of alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen, enzyme-linked immunosorbent assay, we have shown that osthole exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Induction of differentiation by osthole was associated with increased bone morphogenetic protein (BMP)-2 production and the activations of SMAD1/5/8 and p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases. Addition of purified BMP-2 protein did not increase the up-regulation of ALP activity and osteocalcin by osthole, whereas the BMP-2 antagonist noggin blocked both osthole and BMP-2-mediated ALP activity enhancement, indicating that BMP-2 production is required in osthole-mediated osteoblast maturation. Pretreatment of osteoblast cells with noggin abrogated p38 activation but only partially decreased ERK1/2 activation, suggesting that BMP-2 signaling is required in p38 activation and is partially involved in ERK1/2 activation in osthole-treated osteoblast cells. Cotreatment of p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] or p38 small interfering RNA (siRNA) expression inhibited osthole-mediated activation of ALP but only slightly affected osteocalcin production. In contrast, the production of osteocalcin induced by osthole was inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) or by expression of an ERK2 siRNA. These data suggest that BMP-2/p38 pathway links to the early phase, whereas ERK1/2 pathway is associated with the later phase in osthole-mediated differentiation of osteoblast cells. In this study, we demonstrate that osthole is a promising agent for treating osteoporosis.