ABSTRACT
One major problem in cartilage tissue engineering is the insufficient biochemical composition of the generated biocomposites. The aim of this study was to improve the collagen and proteoglycan deposition in tissue engineering constructs by application of long-term mechanical loading in culture. Chondrocyte-seeded chitosan biocomposites revealed a homogenous cell distribution, high viability (>95%) and maintenance of a rounded cell shape typical for chondrocytes over 3 weeks of load-free culture. Cyclic compression of chitosan biocomposites (0.1 Hz, amplitude 5-15%, 45 min on and 315 min off) was applied after two different preculture times (3, 21 days) for 3 weeks. At day 42 this resulted in enhanced mRNA levels for aggrecan and a significantly higher specific proteoglycan (5-fold, p<0.0002) and collagen type II (2-fold, p<0.02) deposition compared to unloaded controls. In sum, the chitosan scaffold was highly attractive for cartilage tissue engineering approaches and mechanical loading allowed to further improve the biochemical composition of these biocomposites.
Subject(s)
Cartilage , Chitosan/metabolism , Chondrocytes/physiology , Tissue Engineering/methods , Animals , Cartilage/chemistry , Chondrocytes/chemistry , Collagen Type II/analysis , Models, Animal , Proteoglycans/analysis , Proteoglycans/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Swine , Time Factors , Weight-BearingABSTRACT
Galantamine (Reminyl), an approved treatment for Alzheimer's disease (AD), is a potent allosteric potentiating ligand (APL) of human alpha 3 beta 4, alpha 4 beta 2, and alpha 6 beta 4 nicotinic receptors (nAChRs), and of the chicken/mouse chimeric alpha 7/5-hydroxytryptamine3 receptor, as was shown by whole-cell patch-clamp studies of human embryonic kidney-293 cells stably expressing a single nAChR subtype. Galantamine potentiates agonist responses of the four nAChR subtypes studied in the same window of concentrations (i.e., 0.1-1 microM), which correlates with the cerebrospinal fluid concentration of the drug at the recommended daily dosage of 16 to 24 mg. At concentrations >10 microM, galantamine acts as an nAChR inhibitor. The other presently approved AD drugs, donepezil and rivastigmine, are devoid of the nicotinic APL action; at micromolar concentrations they also block nAChR activity. Using five CHO-SRE-Luci cell lines, each of them expressing a different human muscarinic receptor, and a reporter gene assay, we show that galantamine does not alter the activity of M1-M5 receptors, thereby confirming that galantamine modulates selectively the activity of nAChRs. These studies support our previous proposal that the therapeutic action of galantamine is mainly produced by its sensitizing action on nAChRs rather than by general cholinergic enhancement due to cholinesterase inhibition. Galantamine's APL action directly addresses the nicotinic deficit in AD.
Subject(s)
Galantamine/pharmacology , Neurons/drug effects , Phenylcarbamates , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolism , Allosteric Regulation , Animals , Carbamates/pharmacology , Cells, Cultured , Cholinesterase Inhibitors/pharmacology , Donepezil , Humans , Indans/pharmacology , Mice , Neurons/metabolism , Piperidines/pharmacology , Receptors, Nicotinic/drug effects , Recombinant Fusion Proteins/drug effects , Rivastigmine , Tacrine/pharmacology , Trichlorfon/pharmacologyABSTRACT
This study analyzes the molecular response of articular chondrocytes to short-term mechanical loading with a special focus on gene expression of molecules relevant for matrix turnover. Porcine cartilage explants were exposed to static and dynamic unconfined compression and viability of chondrocytes was assessed to define physiologic loading conditions. Cell death in the superficial layer correlated with mechanical loading and occurred at peak stresses >or=6 MPa and a cartilage compression above 45%. Chondrocytes in native cartilage matrix responded to dynamic loading by rapid and highly specific suppression of collagen expression. mRNA levels dropped 11-fold (collagen 2; 6 MPa, P=0.009) or 14-fold (collagen 1; 3 and 6 MPa, P=0.009) while levels of aggrecan, tenascin-c, matrix metalloproteinases (MMP1, 3, 13, 14), and their inhibitors (TIMP1-3) did not change significantly. Thus, dynamic mechanical loading rapidly shifted the balance between collagen and aggrecan/tenascin/MMP/TIMP expression. A better knowledge of the chondrocyte response to mechanical stress may improve our understanding of mechanically induced osteoarthrits.
