ABSTRACT
In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.
Subject(s)
DNA Methylation , Single-Cell Analysis , Cell Line , Cell Line, Tumor , CpG Islands , DNA Restriction Enzymes , High-Throughput Screening Assays , Humans , Lymphocytes/metabolism , Polymerase Chain ReactionABSTRACT
One key point for improving osseous integration of implants is to render them osteopromotive by specifically favoring the adhesion of osteoblasts. Mimicking the physiological adhesion process of osteoblasts to the extracellular matrix improves cell adhesion in vitro and results in improved and earlier osseous integration of implants in vivo. Our approach involves coating titanium implants with a tailor-made cyclic-RGD peptide, thus allowing them to bind to specific integrin receptors on the cell surface through multimeric phosphonates. The advantages of this very stable, new type of anchoring for practical application are presented.
Subject(s)
Coated Materials, Biocompatible/chemistry , Oligopeptides/chemistry , Organophosphonates/chemistry , Peptides, Cyclic/chemistry , Prostheses and Implants/trends , Titanium/chemistry , Animals , Cell Adhesion , Coated Materials, Biocompatible/metabolism , Inhibitory Concentration 50 , Integrins/antagonists & inhibitors , Materials Testing , Mice , Molecular Structure , Oligopeptides/pharmacology , Osteoblasts/metabolism , Peptides, Cyclic/pharmacologyABSTRACT
To study the function behind the molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell-adhesive gold nanodots coated with cyclic RGDfK peptide by using block-copolymer micelle nanolithography. The diameter of the adhesive dots is < 8 nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73, and 85 nm spacing at interfaces. A separation of > or = 73 nm between the adhesive dots results in limited cell attachment and spreading, and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in the cell-matrix interface since "micro-nanopatterned" substrates consisting of alternating fields with dense and no nanodots do support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Adhesion/physiology , Integrins/metabolism , Nanotechnology/methods , Cell Adhesion Molecules/chemistry , Cells, Cultured , Fibronectins/chemistry , Gold/chemistry , Integrins/chemistry , Ligands , Micelles , Nanotechnology/instrumentation , Peptides/chemistry , Stress Fibers/metabolismABSTRACT
One keypoint in the development of a biohybrid implant for articular cartilage defects is the specific binding of cartilage cells to a supporting structure. Mimicking the physiological adhesion process of chondrocytes to the extracellular matrix is expected to improve cell adhesion of in vitro cultured chondrocytes. Our approach involves coating of synthetic scaffolds with tailor-made, cyclic RGD-peptides, which bind to specific integrin receptors on the cell surface. In this study we investigated the expression pattern of integrins on the cell surface of chondrocytes and their capability to specifically bind to RGD-peptide coated materials in the course of monolayer cultivation. Human chondrocytes expressed integrins during a cultivation period of 20 weeks. Receptors proved to be functionally active as human and pig chondrocytes attached to RGD-coated surfaces. A competition assay with soluble RGD-peptide revealed binding specificity to the RGD-entity. Chondrocyte morphology changed with increasing amounts of cyclic RGD-peptides on the surface.
Subject(s)
Cartilage, Articular , Cell Adhesion/physiology , Chondrocytes/physiology , Oligopeptides/chemistry , Tissue Engineering/methods , Amino Acid Sequence , Cartilage, Articular/cytology , Cell Adhesion/drug effects , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/drug effects , Humans , Oligopeptides/chemical synthesisABSTRACT
Osteoblasts: yes, platelets: no! Bone implants have to be integrated with the surrounding tissue to allow a smooth and stable connection. A new procedure is shown which is based on covalent linking of a highly selective RGD peptide to a poly(methyl methacrylate) (PMMA) material (see picture). Osteoblasts very effectively bind to the treated surface and are stimulated to proliferate.
