Three-dimensional Migration of Neurites Is Mediated by Adhesion Site Density and Affinity

Abstract

Three-dimensional neurite outgrowth rates within fibrin matrices that contained variable amounts of RGD peptides were shown to depend on adhesion site density and affinity. Bi-domain peptides with a factor XIIIa substrate in one domain and a RGD sequence in the other domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa, and the RGD-dependent effect on neurite outgrowth was quantified, employing chick dorsal root ganglia cultured two-and three-dimensionally within the modified fibrin. Two separate bi-domain peptides were synthesized, one with a lower binding affinity linear RGD domain and another with a higher binding affinity cyclic RGD domain. Both peptides were crosslinked into fibrin gels at concentrations up to 8.2 mol of peptide/mol of fibrinogen, and their effect on neurite outgrowth was measured. Both two-and three-dimensional neurite outgrowth demonstrated a bi-phasic dependence on RGD concentration for both the linear and cyclic peptide, with intermediate adhesion site densities yielding maximal neurite extension and higher densities inhibiting outgrowth. The adhesion site density that yielded maximal outgrowth depended strongly on adhesion site affinity in both two and three dimensions, with lower densities of the higher affinity ligand being required (0.8 -1.7 mol/mol for the linear peptide versus 0.2 mol/mol for the cyclic peptide yielding maximum neurite outgrowth rates in three-dimensional cultures).Cell adhesion and migration in two and three dimensions play important roles in a host of physiological phenomena, including neurite extension (1), angiogenesis (2), and immune responses (3). The adhesion of cells within their environment is controlled primarily by the binding of cell surface receptors to short, exposed protein domains in the extracellular matrix. An important receptor family involved in adhesion and migration is the integrins, a class of heterodimeric receptors that interact with both extracellular matrix and cell-surface ligands (4, 5). Each unique dimeric receptor binds to a particular subset of ligands, which can often be mimicked with immobilized short peptides (6). One such short peptide that binds to integrins and influences subsequent cell adhesion and migration is RGD, a motif present in many proteins, including fibronectin, laminin, collagen, and fibrinogen (7). This sequence has been shown to be recognized by ␣ 5 ␤ 1 , ␣ v ␤ 3 (8), and several other integrin heterodimer pairs.Adhesion and migration upon a surface are distinct processes that depend on related molecular mechanisms. Cell adhesion involves the dynamic interaction between cell surface receptors and adhesion ligands on the supporting matrix (9, 10), with the strength of this attachment being related to the concentration of cell-surface receptors, the concentration of ligands available, and the specific binding affinity of the receptor ligands and pairs (11). Migration of cells upon a material is an analogous phenomenon, with the rate of cell...

Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics

“…Similar to previously published (both experimental and theoretical) reports for cell migration on surfaces (18,32) and within 3D matrices (33)(34)(35)(36) , a biphasic dependence of migration on ligand density was found. In contrast to cell migration across surfaces, 3D cell migration is more complex, as it involves additional cellular strategies to overcome the biophysical resistance of the matrix (37).…”

Nanoscale Adhesion Ligand Organization Regulates Osteoblast Proliferation and Differentiation

“…Overall, the present findings parallel those of many other systems used to present cellular adhesion ligands to cells and probe the effect of ligand types and bulk densities on cell phenotype. 10,11,18,22,23 It was striking that, in certain experiments, the initial spreading of MC3T3 cells was significantly enhanced as the RGD island spacing increased but the growth rate of the cells was unexpectedly suppressed in parallel (Figure 4). These effects were decoupled from the bulk ligand density in the gel ( Figure 5A).…”

Biomimetic Delivery of Keratinocyte Growth Factor upon Cellular Demand for Accelerated Wound Healing in Vitro and in Vivo

“…To do this we conjugated KGF to the peptide (Flc)-LNQEQVSPRKKC, which contains the substrate for factor XIII (NQEQVSP) and can therefore be incorporated into fibrin gels during polymerization. 48,49,56 The first leucine was conjugated to fluorescein to allow quantitation of the bound KGF via fluorescence measurements. The peptide was conjugated to KGF through a cysteine residue that was strategically placed at the end of a highly charged spacer region (RKK).…”

“…46,47 In particular, it was recently demonstrated that bi-domain peptides could be used to covalently attach small cell adhesion peptides 48,49 or heparin-binding growth factors, such as nerve growth factor or brain-derived neurotrophic factor, into fibrin gels. 44 For the latter, one domain of the peptide was recognized by factor XIIIa and incorporated into fibrin during polymerization, while the heparin-binding domain immobilized heparin, which in turn interacted with the heparin-binding growth factor.…”