Dextran has recently been investigated as an alternative to polyethylene glycol (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although anti-fouling properties of ...surface-grafted dextran and PEG are quite similar, the multivalent properties of dextran are advantageous when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired. The preferred methods of dextran immobilization for biomaterial applications should be simple with minimal toxicity. In this report, a method is described for covalent immobilization of dextran to material surfaces which involves low residual toxicity reagents in mild aqueous reaction conditions. 70
kDa MW dextran was immobilized on glass and polyethylene terephthalate (PET) surfaces. 3T3 fibroblast cell adhesion was compared on untreated, aminated, and dextran-coated materials. Dextran coatings effectively limited cell adhesion and spreading on glass and PET surfaces in the presence of serum-borne cell adhesion proteins. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.
The cytotoxicity of polysaccharide-based hydrogels and solutions was studied in vitro after 48h of indirect exposure of the materials with vascular smooth muscle cells. Dextran and/or hyaluronan were ...derivatized using glycidyl methacrylate, and hydrogels were formed in the presence of photoinitiators and ultraviolet radiation in multiwell inserts to avoid direct contact with cell monolayers. Observation of cell morphology indicated that dextran hydrogels, a blend of non-derivatized hyaluronan into dextran hydrogel, and a hyaluronan solution were highly cytocompatible. However, hydrogels made of derivatized hyaluronan were cytotoxic when compared to unexposed sham controls that contained multiwell inserts but no hydrogels. Results from quantitative assays for proliferation and viability corroborated the qualitative observations, and scrape wound assays revealed a significant increase in smooth muscles cell migration/proliferation after indirect exposure to several of the polysaccharide-based materials. Results from this study demonstrate that hydrogels made of dextran and hyaluronan solution show good cytocompatibility in vitro, making these degradable matrices interesting candidates for drug delivery purposes.
We have recently reported the attachment and spreading of human umbilical vein endothelial cells (HUVECs) upon substrates
containing covalently grafted Arg-Glu-Asp-Val (REDV) peptide (Hubbell, J. A., ...Massia, S. P., Desai, N. P., and Drumheller,
P. D. (1991) Bio/Technology 9, 568-572). This peptide has been reported to be the minimal active sequence within the CS5 site
of the alternatively spliced type III connecting segment (IIICS) region of fibronectin, and the integrin alpha 4 beta 1 has
been identified as the receptor on melanoma cells for this site. The integrin alpha 4 beta 1 has also been identified as the
receptor for the CS1 site in the IIICS region on cells of neural crest origin, melanoma cells, lymphocytes, and hematopoietic
stem cells. In this study, we demonstrate that this integrin also serves as a receptor on HUVECs for the peptide REDV from
the CS5 site. The alpha 4 subunit was shown to be expressed upon HUVEC membranes by whole-cell enzyme-linked immunosorbent
assay. Antifunctional antibodies directed against integrin subunits alpha 4 and beta 1 inhibited cell adhesion on REDV-grafted
substrates, but not on RGD-grafted substrates. The alpha 4 subunit localized into fibrillar structures within spread cells
on the REDV-grafted substrates, but not within spread cells on RGD-grafted substrates. Two proteins (144 and 120 kDa) were
isolated from HUVEC extracts by REDV ligand affinity chromatography and were demonstrated by immunoprecipitation and Western
blot to be the integrin subunits alpha 4 (144 kDa) and beta 1 (120 kDa); furthermore, the immunoprecipitation analyses demonstrated
that the subunits formed a complex. HUVEC binding to REDV-grafted substrates was inhibited by both soluble REDV and RGD, demonstrating
that adhesion was biospecific and that the REDV peptide is RGD-like. In this report we demonstrate for the first time that
alpha 4 is present in the endothelial cell membrane, in contrast to previous reports by others, and that integrin alpha 4
beta 1 is the receptor for REDV-mediated adhesion to the IIICS region of region of plasma fibronectin.
We have found a novel adhesion receptor on the human endothelial cell for the peptide sequence Arg-Glu-Asp-Val (REDV), which is present in the III-CS domain of human plasma fibronectin, with a ...dissociation constant of 2.2 x 10(-6) M and 5.8 x 10(6) sites/cell. When a synthetic peptide containing this sequence was immobilized on otherwise cell nonadhesive substrates, endothelial cells attached and spread but fibroblasts, vascular smooth muscle cells, and platelets did not. Endothelial monolayers on REDV were nonthrombogenic: endothelial cells attached and spread upon other receptor-binding domains of fibronectin and laminin, but with lesser degrees of specificity or with a loss of nonthrombogenicity. This approach may provide a basis for a tissue engineered vascular graft where endothelial cell attachment is desired, but not the attachment of other blood vessel wall cells and blood platelets.
The laminin-based nonapeptide Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR) and pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR)
have been previously demonstrated to support the attachment of several cell ...types and to competitively bind to the 67-kDa
high affinity laminin receptor. Cell attachment, but not spreading, on substrates containing adsorbed CDPGYIGSR or YIGSR was
observed. In this report we describe YIGSR-mediated attachment and spreading of a wide variety of cell types. GYIGSRY promoted
cell spreading and stress fiber formation when it was covalently immobilized through the amino-terminal Gly residue, used
as a spacer arm. Spreading was not observed when adsorbed YIGSR peptide was used. Functionally blocking antiserum directed
against the 67-kDa and related laminin-binding proteins blocked human foreskin fibroblast (HFF) spreading, but not attachment,
on covalently grafted GYIGSRY substrates. However, functionally blocking antisera directed against the vitronectin receptor,
integrin alpha v beta 3, and the fibronectin receptor, integrin alpha 5 beta 1, did not affect HFF spreading on these substrates.
When HFFs spread on these substrates, the 67-kDa laminin receptor co-localized with the cytoplasmic proteins alpha-actinin
and vinculin into discrete structures. These results suggest that the adhesion ligand YIGSR is solely sufficient for cell
spreading when it is conformationally constrained by covalent attachment to a solid substrate, at least when attached via
its amino terminus. Furthermore, the role of the 67-kDa laminin receptor in recognition of this ligand and mediating cell
attachment is confirmed in this study. This report also provides the first evidence for direct or indirect association of
this receptor with vinculin and alpha-actinin when YIGSR-mediated cell spreading occurs.
Diamines covalently coupled to glass substrates promoted human foreskin fibroblast adhesion in the absence of serum. These
diamine-derivatized substrates were produced by coupling ethylene diamine, ...N-methylaminoethylamine, and N,N-dimethylaminoethylamine
(NNDMAEA), to sulfonyl chloride-activated glass. Electron spectroscopy for chemical analysis demonstrated that the diamines
were coupled via their primary amine ends to produce a surface-bound secondary amine linked to a free amino moiety via a two-carbon
spacer. NNDMAEA-modified substrates containing free tertiary amines supported the highest degree of cell spreading (73 +/-
7% actively spreading cells) and the most extensive cytoskeletal organization. Both the free tertiary and surface-bound secondary
amines were shown to be required for cell spreading. Lysine- and arginine-grafted substrates supported cell spreading and
cytoskeletal organization similar to that on NNDMAEA-modified substrates. Although some stress fibers were observed within
spread cells on these substrates, focal contacts did not form. Heparinase treatment did not inhibit cell attachment or spreading
to the diamine-derivatized substrates, however chondroitinase ABC inhibited cell attachment and spreading on all substrates;
heparinase inhibited spreading on lysine- and arginine-derivatized substrates to a lesser extent. These results imply that
cell attachment to these substrates was mediated primarily by cell surface chondroitin sulfate proteoglycans. This study demonstrates
that covalently grafted NNDMAEA, lysine, and arginine can mimic the adhesion-promoting activity of the glycosaminoglycan-binding
domains of cell adhesion proteins. This study also demonstrates that the interaction with these proteoglycans depends in a
very sensitive manner on the particular structure of the immobilized amine.
A sol was spun on single crystal silicon substrates at a spin-rate of 3000–5000 rpm followed by a low temperature cure to form a stable sol–gel/silicon structure. Good quality crystalline HA films of ...thickness ∼300–400 nm were obtained by annealing the sol–gel/Si structure in a conventional cavity applicator microwave system with a magnetron power of 1300 W, frequency of 2.45 GHz, and at a low processing temperature of 425 °C for annealing times ranging from 2–60 min. X-ray Diffraction and FTIR analysis confirmed that the crystalline quality of the thin films were comparable or better than those heat-treated under the same processing conditions (temperature and time) in a Rapid Thermal Annealing (RTA) system. The RBS data suggests a composition corresponding to stoichiometric hydroxyapatite Ca₁₀(PO₄)₆(OH)₂, the major inorganic component of bone. The results showed that the HA film thickness decreases with increasing sol spin-rate. The HA films showed good biocompatibility because little monocyte adhesion occurred and hence no inflammatory response was activated in vitro. The potential of microwave annealing for rapid and low temperature processing of good crystalline quality HA thin films derived from sol–gel is demonstrated.
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