Dynamic force spectroscopy to probe adhesion strength of living cells.

We studied the mechanical strength of the adhesion of living cells to model membranes. The latter contained a RGD lipopeptide which is a high affinity binding site for a cell adhesion molecule (integrin alpha(V)beta(3)). Cells adhered specifically to the vesicles. We used micropipette aspiration for breaking this adhesion with well defined forces. Systematic variation of the rate of force application revealed pronounced kinetic effects. The dependence of the detachment forces on the loading rate was well described by a power law (exponent approximately 0.4), in agreement with recent theoretical work.

Decorated surfaces by biofunctionalized gold beads: application to cell adhesion studies.

We describe a simple but versatile method to decorate solid surfaces randomly with colloidal gold particles to which ligands of cell receptors can be coupled to generate local attraction sites for the control of cell adhesion. A self-assembled monolayer of (3-mercaptopropyl)trimethoxysilane was deposited on glass slides. Gold beads were anchored to the functionalized surface through the sulfur group. We characterized the gold bead distribution on the functionalized surface with reflection interference contrast microscopy. The gold beads were functionalized with a disulfide-coupled cyclic pentapeptide containing an arginine-glycine-aspartic acid (RGD) tripeptide sequence which is selectively recognized by integrin receptors alpha(V)beta(3) of endothelial cells. A blocking layer of bovine serum albumin was adsorbed onto the surface to prevent non-specific binding of the cells. We demonstrate that the RGD-functionalized colloidal gold beads act as local attraction centers, mediating rapid cell anchoring on a substrate impeding cell adhesion in the absence of attraction centers. Surprisingly, microinterferometry shows that after a time delay of about 1 h, the regions of the cell surface between the gold beads form close contacts with the substrate, which is attributed to strong van der Waals attraction after escape of repeller molecules from the contact surface.

Selective adhesion of endothelial cells to artificial membranes with a synthetic RGD-lipopeptide.

A constrained cyclic ArgGly-Asp-D-Phe-Lys, abbreviated as cyclo(-RGDfK-), lipopeptide has been synthesized and incorporated into artificial membranes such as giant vesicles with DOPC and solid-supported lipid bilayers. The selective adhesion and spreading of endothelial cells of the human umbilical cord on solids functionalized by membranes with this RGD-lipopeptide have been observed. Furthermore, we have demonstrated strong selective adhesion of giant vesicles to endothelial cells through local adhesion domains by combined application of hydrodynamic flow field and reflection interference contrast microscopy (RICM). The adhesion can be inhibited by competition with a water-soluble RGD peptide. We suggest that this strategy could improve the efficiency of liposomes targeting used as vectors or as drug carriers to cells.

Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation.

The physiological inertness of synthetic implant materials often results in insufficient implant integration and limited acceptance of implants in tissues. After implantation the implant surface is often separated from the surrounding healthy and regenerating tissue, for example by a fibrous capsule. To avoid this host-versus-graft reaction, a strong mechanical contact between tissue and implant must be ensured. An enhanced contact between graft and the surrounding tissue can be provided by coating the implant with cell-adhesive molecules. The highly active and alpha(v)beta(3)- and alpha(v)beta(5)-integrin-selective peptide c(-RGDfK-) (f=D-phenylalanine) was functionalized with various linker molecules containing an acrylamide end group by using the lysine side chain of c(-RGDfK-). The acrylamide group can be used to bind the peptide covalently to poly(methyl methacrylate) (PMMA) surfaces. The coated surfaces effectively bind to murine osteoblasts as well as human osteoblasts in vitro when a minimum distance of 3.5 nm between surface and the constrained RGD sequence is provided. In contrast to osteoblasts in cell suspension, surface-bound osteoblasts show no apoptosis but proliferate by a factor of 10 over a 22 d period. Coating of inert implant surfaces with highly active and alpha(v)-selective peptides affords a marked improvement in osteoblast binding over current technologies. In vivo studies show that peptide-coated PMMA pellets implanted into the patella groove of rabbits are integrated into the regenerating bone tissue faster and more strongly than uncoated pellets.

Induced tissue integration of bone implants by coating with bone selective RGD-peptides in vitro and in vivo studies.

The optimal function of medical implant materials used in tissue substitution is often limited due to its healing properties. This effect is linked to reduced interactions of the implants with the surrounding tissue. Implant surfaces biologically functionalized with arginine-glycine-aspartic acid (RGD) peptides, a class of cellular adhesion factors, are described in this paper. The RGD-peptides are either bound via bovine serum albumin linking on culture plastic dishes as a model surface or via acrylic acid coupling on PMMA surface as a potential implant material. Resulting functionalized surfaces aquire the capability to bind cultured osteoblasts in high levels and show high proliferation rates in vitro. These results are observed for osteoblast cultures as well as from different species with different preparation procedures. A critical minimum distance between the bioactive portion of the RGD-peptides and the implant surface of 3.0-3.5 nm is crucial for the induction of an optimum cell binding process. In vivo animal studies in the rabbit show that newly formed bone tissue generated a direct contact with the RGD-peptide coated implants. In contrast uncoated implants are separated from newly formed bone tissue by a fibrous tissue layer thereby preventing the formation of a direct implant-bone bonding.