-
[show abstract]
[hide abstract]
ABSTRACT: In medicine, N-methyl pyrrolidone (NMP) has a long track record as a constituent in medical devices approved by the Food and Drug Administration and thus can be considered as a safe and biologically inactive small chemical. In the present study, we report on the newly discovered pharmaceutical property of NMP in enhancing bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with bone morphogenetic protein (BMP)-2, because NMP increased early and late markers for maturation of preosteoblasts and human bone marrow-derived stem cells in vitro. When we used the multipotent cell line C2C12 without autologous BMP expression, NMP alone had no effect on alkaline phosphatase activity, a marker for osteogenic transdifferentiation. Nevertheless, in combination with low BMP-2 doses, alkaline phosphatase activity was more than eight times as great. Thus, the pharmaceutical NMP mode of action is that of an enhancer of BMP activity. The dependency of the effects of NMP on BMP was confirmed in preosteoblasts because noggin, an extracellular BMP inhibitor, suppressed NMP-induced increases in early markers for osteoblast maturation in vitro. At the molecular level, NMP was shown to have no effect on the binding of BMP-2 to the ectodomain of the high-affinity BMP receptor IA. However, NMP further increased the phosphorylation of p38 and Smad1,5,8 induced by BMP-2. Thus, the small chemical NMP enhances BMP activity by increasing the kinase activity of the BMP receptor complex for Smad1,5,8 and p38 and could be employed as a potent drug for bone tissue regeneration and engineering.
Tissue Engineering Part A 04/2009; 15(10):2955-63. · 4.64 Impact Factor
-
Andreas H Zisch,
Matthias P Lutolf,
Martin Ehrbar,
George P Raeber,
Simone C Rizzi,
Neil Davies, Hugo Schmökel,
Deon Bezuidenhout,
Valentin Djonov,
Peter Zilla,
Jeffrey A Hubbell
[show abstract]
[hide abstract]
ABSTRACT: Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.
The FASEB Journal 01/2004; 17(15):2260-2. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or have undergone total hip replacement. In this report, stable folding variants of the natural occurring osteoinductive BMPs were shown to act as inhibitors for heterotopic ossification. The most effective BMP folding variant construct performed even better than the natural occurring BMP antagonist Noggin because it also inhibited calcium deposition of pre-osteoblastic cells.
Signal transduction through receptor and ligand binding depends on the proper folding of all partners, especially when it involves the formation of a heterotetramer. In the case, the receptor binding of the ligand can be uncoupled from signal transduction, and folding variants of a ligand can be developed into antagonists of the natural bioactivity of the ligand. Here we present a deletion mutant of a bone morphogenetic protein (BMP) folding variant capable of inhibiting the bone-inducing action of natural occurring BMPs.
Deletion mutants and site-directed mutants of BMP folding variants were generated and tested for their ability to reduce alkaline phosphatase activity and mineralization in a pre-osteoblastic cell line. In vivo activity of the optimized folding variant was determined in a heterotopic ossification model in rodents and in two Xenopus laevis model systems. Biosensor interaction analysis was used to determine the affinity of the optimized BMP folding variant to the extracellular domain of BMP receptors.
In vitro and in vivo tests in rodents revealed that the structural elements of the wrist epitope combined with finger 2 and a positive charge proximal to the tip of this finger are sufficient to induce osteoinhibition with deletion mutants and folding variants of mature BMP-4. The inhibitor designed to suppress heterotopic ossification showed BMP antagonist activity in embryos and animal caps of X. laevis. Binding studies of the inhibitor to ectodomains of type I and type II BMP receptors revealed a concentration-dependent binding, especially to the high-affinity BMP receptor.
Deletion mutants of BMP folding variants are a new form of BMP antagonists and act through competition with osteoinductive BMP for BMP receptor binding. The excellent in vivo performance of the optimized folding variant is because of its ability to block signaling of endogenous BMPs deposited in the extracellular matrix even more effectively than the natural occurring BMP antagonist Noggin.
Journal of Bone and Mineral Research 01/2004; 18(12):2142-51. · 6.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Graft copolymers were designed that could spontaneously bind to biological surfaces and block subsequent recognition and adhesion at those surfaces. Phenylboronic acid (PBA) moieties in the polymer backbone provided binding to surfaces, forming reversible covalent complexes with cis-diols found in many biological molecules. Pendant poly(ethylene glycol) (PEG) side chains sterically protected those surfaces from subsequent interactions with other proteins and cells. The PEG and PBA grafting ratios on these poly-L-lysine-graft-(PEG;PBA) copolymers [PLL-g-(PEG;PBA)] were varied, and the polymers were tested in models relevant to undesirable wound-healing responses such as peritoneal adhesion formation and posterior capsule opacification. PLL-g-(PEG;PBA) polymers spontaneously coated tissue culture polystyrene and completely blocked rabbit lens epithelial cell adhesion to the surface over a wide range of PEG grafting ratios. PLL-g-(PEG;PBA)s with optimal grafting ratios were able to coat adsorbed serum proteins or extracellular matrices and block cell spreading on the surfaces at 4 h, although the effect was lost within 24 h. The polymer also enhanced the efficacy of surgical lysis of peritoneal adhesions in rats. The reversible covalent complexes formed by the PBA moieties on the copolymer backbone were more effective at binding biological surfaces than electrostatic interactions formed via a copolymer lacking the PBA moieties, that is, PLL-g-PEG.
Journal of Biomedical Materials Research 04/2002; 59(4):618-31.
