sepharose and arginyl-glycyl-aspartic-acid

Our previous reports of interpenetrating networks (IPNs) have demonstrated drastic improvements in mechanical performance relative to individual constituent networks while maintaining viability of encapsulated cells. The current study investigated whether covalent linkage of RGD to the poly(ethylene glycol) diacrylate (PEGDA) network could improve upon cell viability and performance of agarose-PEGDA IPNs compared to unmodified IPNs (control) and to IPNs with different concentrations of physically entrapped aggrecan, providing a point of comparison to previous work. The inclusion of RGD or aggrecan generally did not adversely affect mechanical performance, and significantly improved chondrocyte viability and performance. Although both 4 and 100 μg/mL of aggrecan improved cell viability, only 100 μg/mL aggrecan was clearly beneficial to improving biosynthesis, whereas 100 μg/mL of RGD was beneficial to both chondrocyte viability and biosynthesis. Interestingly, clustering of cells within the IPNs with RGD and the higher aggrecan concentration were observed, likely indicating cell migration and/or preferred regional proliferation. This clustering resulted in a clearly visible enhancement of matrix production compared to the other IPNs. With this cell migration, we also observed significant cell proliferation and matrix synthesis beyond the periphery of the IPN, which could have important implications in facilitating integration with surrounding cartilage in vivo. With RGD and aggrecan (at its higher concentration) providing substantial and comparable improvements in cell performance, RGD would be the recommended bioactive signal for this particular IPN formulation and cell source given the significant cost savings and potentially more straightforward regulatory pathway in commercialization.

Topics: Aggrecans; Animals; Biocompatible Materials; Cattle; Cell Proliferation; Cell Shape; Cell Survival; Chondrocytes; DNA; Extracellular Matrix; Glycosaminoglycans; Hydrogels; Hydroxyproline; Immobilized Proteins; Magnetic Resonance Spectroscopy; Male; Mechanical Phenomena; Oligopeptides; Polyethylene Glycols; Sepharose; Spectroscopy, Fourier Transform Infrared; Sus scrofa

To obtain sufficient cell numbers for cartilage tissue engineering with autologous chondrocytes, cells are typically expanded in monolayer culture. As a result, they lose their chondrogenic phenotype in a process called dedifferentiation, which can be reversed upon transfer into a 3D environment. We hypothesize that the properties of this 3D environment, namely adhesion site density and substrate elasticity, would influence this redifferentiation process. To test this hypothesis, chondrocytes were expanded in monolayer and their phenotypical transition was monitored. Agarose hydrogels manipulated to give different RGD adhesion site densities and mechanical properties were produced, cells were incorporated into the gels to induce redifferentiation, and constructs were analyzed to determine cell number and extracellular matrix production after 2 weeks of 3D culture. The availability of adhesion sites within the gels inhibited cellular redifferentiation. Glycosaminoglycan production per cell was diminished by RGD in a dose-dependent manner and cells incorporated into gels with the highest RGD density, remained positive for collagen type I and produced the least collagen type II. Substrate stiffness, in contrast, did not influence cellular redifferentiation, but softer gels contained higher cell numbers and ECM amounts after 2 weeks of culture. Our results indicate that adhesion site density but not stiffness influences the redifferentiation process of chondrocytes in 3D. This knowledge might be used to optimize the redifferentiation process of chondrocytes and thus the formation of cartilage-like tissue.

Topics: Animals; Cattle; Cell Adhesion; Cell Count; Cell Culture Techniques; Cell Dedifferentiation; Cell Differentiation; Cell Proliferation; Chondrocytes; Collagen Type I; Collagen Type II; Elasticity; Hydrogels; Immunohistochemistry; Oligopeptides; Sepharose; Serum Albumin, Bovine; Sus scrofa

Cells actively probe the stiffness of their surrounding and respond to it. The authors recently found that maintenance of the chondrogenic phenotype was directly influenced by this property in 2D. Since studies about this process in 3D are still largely absent, this study aimed to transfer this knowledge into a 3D environment. Agarose was modified with RGD to allow active stiffness sensing or RGE as a control. Hydrogels with different mechanical properties were produced by using different concentrations of agarose. Primary chondrocytes were incorporated into the gel, cultured for up to two weeks, and then constructs were analyzed. Cells were surrounded by their own ECM from an early stage and maintained their chondrogenic phenotype, independent of substrate composition, as indicated by a high collagen type II and a lack of collagen type I production. However, softer gels showed higher DNA and GAG content and larger cell clusters than stiff gels in both RGD- and RGE-modified agarose. The authors hypothesize that matrix elasticity in the tested range does not influence the maintenance of the chondrogenic phenotype in 3D but rather the size of the formed cell ECM clusters. The deviation of these findings from previous results in 2D stresses the importance of moving towards 3D systems that more closely mimic in vivo conditions.

Topics: Animals; Cells, Cultured; Chondrocytes; Collagen Type I; Collagen Type II; Elasticity; Extracellular Matrix; Hydrogels; Oligopeptides; Sepharose; Swine

Thermoresponsive and injectable semi-interpenetrating polymer networks (sIPNs) containing a biospecific cell-adhesive signal and proteolytically degradable domains were developed as a synthetic equivalent of the extracellular matrix (ECM). The sIPNs synthesized define a modular hydrogel ECM where different properties of the matrix can be manipulated independently, thus creating a system where parametric analysis of the effect of hydrogel properties on cell proliferation and differentiation is possible. sIPNs composed of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)] and RGD-grafted poly(acrylic acid) linear chains [p(AAc)-g-RGD] were synthesized with peptide crosslinkers containing a matrix metalloproteinase-13 (MMP-13, collagenase-3) degradable domain. The lower critical solution temperature (LCST) of peptide-crosslinked p(NIPAAm-co-AAc) sIPNs was not influenced by the addition of either linear p(AAc) or peptide-modified p(AAc) chains ( approximately 34 degrees C) in PBS. Degradation of peptide-crosslinked hydrogels and sIPNs was enzyme specific and concentration dependent. Exposure of rat calvarial osteoblast (RCO) culture to the degradation products from the peptide-crosslinked hydrogels did not significantly affect cell viability. Migration of RCOs into the sIPNs was dependent upon the presence of both a cell-adhesive RGD peptide (Ac-CGGNGEPRGDTYRAY-NH2) and proteolytically-degradable crosslinks; however, there was greater dependence on the latter. The sIPNs synthesized are versatile materials for assessing cell fate in synthetic ECM constructs in vitro and tissue regeneration in vivo.

Topics: Acrylamides; Acrylates; Animals; Biocompatible Materials; Cell Adhesion; Cell Differentiation; Cell Lineage; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Collagenases; Cross-Linking Reagents; Extracellular Matrix; Hot Temperature; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Mass Spectrometry; Matrix Metalloproteinase 13; Microscopy, Phase-Contrast; Models, Chemical; Oligopeptides; Osteoblasts; Peptides; Polymers; Protein Structure, Tertiary; Rats; Regeneration; Sepharose; Temperature; Time Factors; Tissue Engineering

Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A(12) infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.

Topics: Amino Acid Sequence; Animals; Binding Sites; Capsid Proteins; Cell Line; China; Cricetinae; Foot-and-Mouth Disease; Foot-and-Mouth Disease Virus; Genetic Engineering; Heparitin Sulfate; Models, Molecular; Molecular Sequence Data; Oligopeptides; Recombinant Fusion Proteins; Sepharose; Swine; Swine Diseases; Virion

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20 degrees -37 degrees C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells.

Topics: Blood Vessel Prosthesis; Carbon Dioxide; Cell Culture Techniques; Cell Division; Cells, Cultured; Culture Media; Endothelium, Vascular; Fibroblast Growth Factor 2; Fibronectins; Heparin; Humans; Iodine Radioisotopes; Microspheres; Oligopeptides; Polystyrenes; Receptors, Fibronectin; Receptors, Vitronectin; Recombinant Fusion Proteins; Sepharose; Serum Albumin; Sterilization; Sulfur Radioisotopes

A procedure for the isolation of osteopontin (OPN) from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded approximately 8 mg of purified protein per liter of milk. SDS-PAGE analysis revealed that the protein migrated at M(r) 60,000. NH2-terminal sequence analysis of the first seven amino acids revealed the protein to be identical to that previously reported for bovine OPN. Also, our preparation demonstrated expected biological properties of OPN including adhesion of both endothelial and vascular smooth muscle cells to OPN in a dose- and Arg-Gly-Asp-dependent manner. Furthermore, OPN coupled to Sepharose was capable of binding the alpha v beta 3 integrin from a detergent extract of endothelial cells. Thus, our procedure yielded biologically active OPN from an abundant and natural source.

Topics: Amino Acid Sequence; Animals; Cattle; Cell Adhesion; Cells, Cultured; Chromatography; Chromatography, Agarose; Chromatography, Ion Exchange; Endothelium, Vascular; Female; Humans; Milk; Molecular Weight; Muscle, Smooth, Vascular; Oligopeptides; Osteopontin; Rats; Receptors, Vitronectin; Sepharose; Sialoglycoproteins

Myelination of the CNS requires the migration of oligodendrocyte precursors throughout the CNS from restricted regions within the ventricular and subventricular zones. In light of the significant effects of cell-extracellular matrix (ECM) interactions on cell migration in other developing systems, we have analyzed the role of integrins in oligodendrocyte precursor migration. We have shown previously that oligodendrocyte precursors in vitro express a limited repertoire of integrins, including alpha 6 beta 1, alpha v beta 3, and that differentiation is associated with downregulation of alpha v beta 1 and upregulation of alpha v beta 5. Using a migration assay based on the movement of cells away from an agarose drop containing a high-density cell suspension, we find that RGD peptides (that block alpha v but not alpha 6 integrins) and anti-beta 1 antibodies block migration on an astrocyte-derived ECM, whereas anti-beta 3 antibodies have little effect. These results suggest that alpha v beta 1 but not alpha 6 beta 1 plays a role in oligodendrocyte precursor migration, and this is confirmed by the use of blocking monoclonal antibodies that distinguish these two integrins. In keeping with the results of others, we find that differentiated oligodendrocytes lose migratory potential and that the timing of this loss correlates with downregulation of alpha v beta 1. Taken together with the work of others showing that ECM ligands for alpha v beta 1 are expressed within the CNS, we propose that this integrin plays a significant role in the migration of oligodendrocyte precursors in vivo and that its downregulation during differentiation could be an important factor regulating the migratory phenotype of these cells.

Topics: Animals; Animals, Newborn; Antibody Specificity; Astrocytes; Cell Differentiation; Cell Movement; Collagen; Extracellular Matrix; Fibronectins; Integrin alpha6beta1; Integrins; Laminin; Mice; Oligodendroglia; Oligopeptides; Platelet-Derived Growth Factor; Rats; Receptors, Vitronectin; Sepharose; Stem Cells; Vitronectin

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*-peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.

Topics: Amino Acid Sequence; Animals; Antibody Specificity; Bacteriophages; Binding Sites; Binding, Competitive; Cell Adhesion; Fibronectins; Gene Library; Humans; Immunoblotting; Integrins; Mice; Mice, Inbred Strains; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptides; Sepharose

We have described previously an adhesive protein on neutrophils (PMN) which recognizes fibrinogen, fibronectin (Fn), von Willebrandt's factor, vitronectin, collagen, and synthetic peptides containing the Arg-Gly-Asp (RGD) sequence (Gresham et al., J. Cell Biol. 108, 1935-1943, 1989). We have called this oligospecific receptor the leukocyte response integrin (LRI). Engagement of LRI leads to both increased ingestion via PMN IgG Fc receptors and to adhesion and chemotaxis to certain extracellular matrix proteins. Now, we have purified an RGD-binding receptor from DMSO-differentiated HL-60 cells (dHL-60) by peptide affinity chromatography which has the biochemical, immunologic, and functional characteristics of LRI. The purified protein contains two bands of 135 and 90 kDa under nonreducing conditions SDS-PAGE. Immunologic characterization of the dHL-60 RGD receptor showed that, by Western blot and ELISA, the lower M(r) band was recognized by mAb 7G2, raised against placental beta 3, which is known to inhibit LRI function. However, despite this functional and immunologic cross-reactivity with beta 3, the receptor was not recognized efficiently by a polyclonal antibody to placental RGD-binding proteins, predominantly alpha v beta 3. Moreover, polyclonal antibody raised to the dHL-60 receptor (Ab1) did not react with placental RGD-binding proteins. By immunoprecipitation or ELISA, we demonstrated that the purified RGD-binding receptor was not alpha IIb beta 3 or alpha v beta 3 and did not contain the integrin chains alpha 4, beta 2, or beta 7. Functionally, Ab1 totally inhibited Fn-stimulated ingestion by PMN. Moreover, Ab1 inhibited phagocytosis stimulated by the peptide KGAGDV, which is the most specific ligand for LRI currently known, and Ab1 inhibited the binding of KGAGDV-coated microspheres to PMN and monocytes. FACS analysis with Ab1 showed staining of monocytes, PMN, and lymphocytes but not platelets or erythrocytes. We conclude that LRI is a novel RGD-binding receptor which exists on leukocytes and which shares an antigenic epitope(s) with beta 3. This receptor recognizes multiple RGD-containing ligands and can mediate signal transduction for adhesion, chemotaxis, and activation of increased phagocytic potential by PMN and monocytes.

Topics: Amino Acid Sequence; Antibodies; Antibodies, Monoclonal; Blood Cells; Extracellular Matrix Proteins; Humans; Integrins; Leukemia, Myelomonocytic, Acute; Macromolecular Substances; Molecular Sequence Data; Neutrophils; Oligopeptides; Phagocytes; Phagocytosis; Protein Binding; Receptors, Immunologic; Receptors, Peptide; Sepharose