Four plasma proteins have been shown to be able to mediate platelet adhesion to synthetic materials when they are adsorbed as purified proteins: fibrinogen (Fg), fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWF). Among them, Fg is thought to play a leading role in mediating platelet adhesion to plasma-preadsorbed biomaterials, but this has been established for only three types of materials so far in our laboratory. Furthermore, the role of Fn, Vn, and vWF in mediating platelet adhesion to plasma-preadsorbed surfaces is still unclear. The aim of the current study was to assess the importance of Fg, Fn, Vn, and vWF in mediating platelet adhesion to a series of polystyrene-based surfaces. The strategy applied in the present investigation was to compare platelet adhesion to surfaces preadsorbed with normal plasma, plasma selectively depleted in Fn or Vn or both Fn and Vn, plasma from donors who were genetically deficient in vWF, and serum. Few platelets adhered to the surfaces preadsorbed with serum, whereas depletion of Fn, Vn, or vWF from plasma did not decrease platelet adhesion significantly. Replenishment of exogenous Fg to serum before protein adsorption restored platelet adhesion to the surfaces, suggesting that Fg was the major plasma protein that mediated platelet adhesion. Also, we found that a surface density of adsorbed Fg far below the amount that usually adsorbs to synthetic surfaces was sufficient to support full-scale platelet adhesion.

No abstract

The ability to create thick tissues is a major tissue engineering challenge, requiring the development of a suitable vascular supply. Current trends are seeing the utilization of cells seeded into hybrid matrix/scaffold systems to create in vitro vascular analogues. Approaches that aim to create vasculature in vitro include the use of biological extracellular matrices such as collagen hydrogels, porous biodegradable polymeric scaffolds with macro-and micro-lumens and micro-channels, co-culture of cells, incorporation of growth factors, culture in dynamic bioreactor environments, and combinations of these. Of particular interest are those approaches that aim to create bioengineered tissues in vitro that can be readily connected to the host's vasculature following implantation in order to maintain cell viability.

To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bone-derived cells (HBDC) to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS). At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin (Vn) from serum onto the EDS domains. In contrast, fibronectin (Fn) was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. In this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function.

In recent years a central objective of tissue engineering has been understanding the interaction of cells with biomaterial surfaces. In this study we examined the protein adsorption events necessary to control the attachment and the subsequent spatial distribution of bone-derived cells exposed to chemically modified surfaces. Silane chemistry and photolithography techniques were used to create substrates with alternating regions of an aminosilane, N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS), along side an alkylsilane, dimethyldichlorosilane (DMS), on quartz surfaces. Sera depleted of fibronectin (Fn), vitronectin (Vn), or both were used to determine if these proteins were necessary for the initial attachment and spatial distribution of bone-derived cells exposed to modified surfaces in vitro. The kinetics and mechanisms of the spatial distribution of cells were examined using light microscopy and digital image acquisition and subsequently were analyzed. Compared to complete serum, the use of serum depleted of fibronectin with vitronectin included had minimal effect on the cell attachment, spreading, and spatial distribution on the EDS regions of the surface. However, the use of serum depleted of vitronectin with or without fibronectin included resulted in greatly reduced cell attachment and spreading. Thus the presence of vitronectin was required for the attachment, spreading, and spatial distribution of bone-derived cells exposed to EDS/DMS-patterned surfaces.