Microcapsules made by enzymatically tailored alginate

Alginate is widely used for encapsulation of cells. Alginate is a linear block copolymer consisting of mannuronic acid (M) and guluronic acid (G). It has been shown that enzymes known as C-5 epimerases convert M to G in the polymer chain, giving rise to novel alginates with tailored properties. One of these enzymes, AlgE4, converts M blocks into blocks of strictly alternating M and G. In this study we investigated how alginate epimerized by AlgE4 affected capsule properties such as stability and permeability. Inhomogeneous calcium-alginate gel beads were made with original and AlgE4-epimerized alginates of different origin. The epimerized alginates formed initially smaller alginate gels that showed increased resistance to osmotic swelling compared with the original nonmodified alginate samples. The permeability, measured as diffusion of immunoglobulin (Ig) G into Ca/Ba-alginate gel beads, was reduced by epimerization and further reduced by addition of poly-L-lysine (PLL). The osmotic stability of alginate-poly-D-lysine(PDL)-alginate capsules was enhanced by the use of epimerized alginate; indeed, stable capsules with low permeability to tumor necrosis factor (TNF) could be made with low PDL exposures. Finally, alginate with an alternating structure interacted more strongly with the alginate-PLL capsule than did alginate with a high content of M blocks or G blocks or than an alginate consisting mainly of M.     

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.     

Evidence of crosslinks in a butadiene-divinylbenzene-ethylvinylbenzene copolymer by metathesis degradation

1,3-Butadiene was copolymerized with a mixture of divinylbenzene isomers containing ethyl-vinylbenzene isomers. The crosslinked copolymers were degraded in a cross metathesis reaction with (Z)-2-butene or (E)-3-hexene using the catalyst WCl₆/Sn(CH₃)₄. The low-molecular-weight products were investigated by gas chromatography/mass spectrometry coupling. Products containing the crosslinks were found. Others were attributable to copolymer units resulting from butadiene and ethylvinylbenzene. Characteristic mass spectra of the products are discussed.     

Surface functionalization of degradable polymers by covalent grafting

With a new non-destructive and solvent-free photografting technique, N-vinylpyrrolidone was covalently grafted onto the surfaces of degradable polymers; poly(l-lactide), poly(epsilon-caprolactone), poly(lactide-co-glycolide), and poly(trimethylene carbonate). The modified surfaces were characterized by XPS, ATR-FTIR, SEM, and cell growth tests. The wettability was markedly improved, as static contact angles changed from about 80 degrees for the pristine substrates to around 30 degrees after 30min of grafting. Well-defined surface topographies, such as micro-patterns, are preserved in the process since the graft layers are thin. The biological response, measured as cytotoxicity, showed that the modified films provide good substrates, comparable with optimized cell culture plastics, for the adhesion and proliferation of normal human keratinocytes and skin fibroblasts.     

Inhibition of lymphocyte-induced angiogenesis by enzymatically isolated rabbit cornea cells.

Corneal and kidney cells were isolated from adult rabbits by enzymatic digestion. The were tested for anti-angiogeneic activity by the lymphocyte-induced angiogenesis assay. In this assay an intradermal injection of semi-allogeneic lymphocytes resulted in a new blood vessel formation visible after three days at the injection site. Isolated rabbit cells were mixed in 1:10 ratio with murine lymphocytes and injected into 600 R X-ray irradiated mice. Number of newly formed blood vessels evoked by lymphocytes injected alone or with rabbit cells added was counted. Corneal cells but not kidney cells decreased angiogenesis evoked by lymphocytes. This finding is discussed in view of corneal avascularity and pathological neo-vascularization.     

Polymers containing enzymatically degradable bonds V. Hydrophilic polymers degradable by papain

Copolymers of N-(2-hydroxypropyl)methacrylamide were prepared, in which synthetic polymer chains are joined by crosslinks containing oligopeptidic sequences degradable with papain, with the general structure P-(Gly)_nX-Y-NH-(CH₂)₆6-NH-Y-X-(Gly)_n-P (P is the polymer chain, N = 1, 2; X…Phe, Val, Gly;Y... Lys, Gly, Tyr, Ala, Phe). The relationship between the structure of these polymeric substrates and their degradability with papain was investigated viscometrically. It was shown that -Phe-Lys- was the most suitable -X-Y- sequence. Extension of the oligopeptidic sequence by one amino acid residue causes a pronounced rise in the rate of cleavage of the polymeric substrates.     

Physical and biological evaluations of sintered hydroxyapatite/silicone composite with covalent bonding for a percutaneous implant material

A composite (HA/silicone) of hydroxyapatite (HA) microparticles with an average diameter of 2.0 micro m covalently linked to a silicone substrate has been developed, and its physical and biological properties as a percutaneous soft-tissue-compatible material have been evaluated. In tensile property measurement, samples of HA/silicone and the original silicone were similar in tensile strength, ca. 7.8 MPa, and elongation at break, ca. 570%. It was found that chemical surface modification with HA particles presented no mechanical disadvantage. In an adhesive-tape peeling test, scanning electron microscopic (SEM) observation showed that HA particles coupled directly to the substrate were not removed. HA particles may bond strongly with the substrate. In human periodontal ligament fibroblast attachment and proliferation experiments, the number of cells attached to HA/silicone was 14 times greater than that attached to the original silicone after 24 h of incubation. The value on HA/silicone was ca. 80% versus that on a tissue-culture plastic used as a positive control. After 72 h of incubation, the number of cells grown on HA/silicone increased to the level of the positive control. In observation of fluorescence microscopy stained by Hoechst 33342, cells appeared to tightly adhere to HA particles coupled to the silicone sheet due to intact nuclear morphology. Observation of cells by fluorescence dye with rhodamin phalloidin showed an extensive F-actin cytoskeleton on HA/silicone. In a 4-week animal implant test, force required to pull out the HA/silicone sheet was 15 times that of the original silicone. HA-particle coating on silicone with covalent linkage gave the inert surface bioactivity. The HA composite thus effectively prevents germ infection percutaneously.     

Heritable activity: a prion that propagates by covalent autoactivation

Known prions (infectious proteins) are self-propagating amyloids or conformationally altered proteins, but in theory an enzyme necessary for its own activation could also be a prion (or a gene composed of protein).We show that yeast protease B is such a prion, called [beta].[beta] is infectious, reversibly curable, and its de novo generation is induced by overexpression of the pro-protease. Present in normal cells but masked by the functionally redundant protease A, [beta] is advantageous during starvation and necessary for sporulation.We propose that other enzymes whose active, modified, form is necessary for their maturation might also be prions.     

Polymers containing enzymatically degradable bonds. VI.Hydrophilic gels cleavable by chymotrypsin

New types of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide which contain oligopeptide sequences in the crosslinks were prepared. These gels are enzymatically degradable by chymotrypsin. The rate of their degradation may be varied within a broad range by changes in the length and detailestructure of the oligopeptide sequence in the crosslinks and by changing their network density.     

Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically tailored pectins

Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR-alpha and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR-alpha did not. On MHR-alpha, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications.     

Increased diversity of intestinal antimicrobial peptides by covalent dimer formation

Antimicrobial peptides are essential effector molecules of the innate immune system. Here we describe the structure, function and diversity of cryptdin-related sequence (CRS) peptides, a large family of antimicrobial molecules. We identified the peptides as covalent dimers in mouse intestinal tissue in amounts comparable to those of Paneth cell-derived enteric alpha-defensins. CRS peptides caused rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides formed homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract.     

Apparent movements induced by stroboscopic illumination of stabilized images

Summary Stroboscopic illumination of stabilized images causes habituation effects and illusory phenomena which, at low stimulation frequencies, show up as brighter patches. When a line is used as a stimulus, these patches are observed as brighter spots in the line surrounded by dimmer patches in the background. These locally brighter parts function as sources from which brightness spreads in the direction of the line at light-on and as sinks to which the brightness retracts again after light-off. This spread and retraction of the brightness induces the perception of movements.Higher stimulation frequencies or a diminished stimulus-background contrast enables the brightness to spread also in the direction perpendicular to the line contour, i.e., into the background. Again a perception of movement is induced, local displacements of a part of the field are observed as a result of the brightness spread.Tiny pinpoints of light, the smallest foveal perceptive elements, visible in lines narrower than 10 min arc (Gerrits, 1978), are also observed when these lines are illuminated with Stroboscopic light. These tiny elements do not spread their brightnesses and so also no movement is induced.The results are discussed in relation to the properties of the perceptive elements and the cells activating them (habituation, barriers, filling-in).Part of this work has been presented on an International Workshop on Spatial Contrast in Amsterdam (The Netherlands) in January 1976 and on the Summer School on Integration of Sensory Information in Erice on Sicily (Italy) from April 19 to May 2, 1976R. van Domburgh participated as a student in these investigations in the last two years before the end of his study     

Functional CD1a is stabilized by exogenous lipids

Self-glycosphingolipids bind to surface CD1 molecules and are readily displaced by other CD1 ligands. This capacity to exchange antigens at the cell surface is not common to other antigen-presenting molecules and its physiological importance is unclear. Here we show that a large pool of cell-surface CD1a, but not CD1b molecules, is stabilized by exogenous lipids present in serum. Under serum deprivation CD1a molecules are altered and functionally inactive, as they are unable to present lipid antigens to T cells. Glycosphingolipids and phospholipids bind to, and restore functionality to CD1a without the contribution of newly synthesized and recycling CD1a molecules. The dependence of CD1a stability on exogenous lipids is not related to its intracellular traffic and rather to its antigen-binding pockets. These results indicate a functional dichotomy between CD1a and CD1b molecules and provide new information on how the lipid antigenic repertoire is immunologically sampled.     

Crosslink formation in porcine valves stabilized by dye-mediated photooxidation

Bovine pericardial and porcine valve materials stabilized by dye-mediated photooxidation have shown potential for bioprosthetic valve use. Previously, in vitro and in vivo stability of these materials was demonstrated through enzymatic, chemical, extraction, rat subcutaneous, and functional challenges. Here, we examine the stability of photooxidized porcine aortic valves through amino acid, crosslink, and hydrothermal isometric tension analysis. Photooxidation reduced intact histidine residues from 17.0 to 0 residues per 1000, indicating the photooxidative alteration of this amino acid. Diphenyl borinic acid-derivitized hydrolyzates of proteins were separated by high-performance liquid chromatography, which identified several amino acid crosslinks that appeared with photooxidation that were absent in untreated controls. Thermal relaxation analysis indicated a significantly higher (p < 0.0002) thermal stability for photooxidized porcine cusps than that of untreated controls, with mean relaxation times for untreated cusps of 14,000 +/- 4650 versus 22,900 +/- 2480 s for photooxidized cusps. In summary, porcine aortic valve tissue treated by dye-mediated photooxidation contains new chemical species and exhibits properties consistent with intermolecular crosslink formation, which explain the increased biostability of this material and its potential for use in bioprosthetic devices.     

Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins

The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-α) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24 h on MHR-α-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-α secretion were studied. The results indicate that MHR-α coating inhibits the LPS-induced activation of macrophages.     

Immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding

We developed a new biomaterial effective for nerve regeneration consisting of molecularly aligned chitosan with laminin peptides bonded covalently. Molecularly aligned chitosan was prepared from crab (Macrocheira kaempferi) tendons by ethanol treatment and 4 wt%-NaOH aqueous solutions to remove proteins and calcium phosphate, followed by deacetyl treatment using a 50 wt%-NaOH aqueous solution at 100 degrees C. Molecularly aligned tendon chitosan was chemically thiolated by reacting 4-thiobutyrolactone with the chitosan amino group. The introduction of thiol groups and their distribution to tendon chitosan and chitosan cast film were confirmed using ATR FT-IR, (1)H-NMR, and EDS. The 1.24 micromol/g of thiol groups introduced on the surface of tendon chitosan and the chitosan cast film was confirmed using ultraviolet (UV) spectra. Thiol groups of cysteine located at the end of synthetic laminin peptides were then reacted chemically with thiolated chitosan to form chitosan-S-S-laminin peptide. YIGSR estimated at 0.92 micromol/g and IKVAV estimated at 0.28 micromol/g on thiolated tendon chitosan were confirmed using UV spectra. YIGSR was estimated at 0.85 micromol/g and IKVAV was estimated at 0.34 micromol/g on the thiolated chitosan cast film.     

New multiphase thermoplastic elastomers by combination of covalent and association-chain structures

A new type of multicomponent thermoplastic elastomers is prepared by attaching 4-(3,5-dioxo-1,2,4-triazolin-4-yl)benzoic acid to polybutadiene chains. The groups are difunctional with respect to a potential chain-molecule formation via reversible association. Thus, a covalent polymer is combined with an association polymer. Characterisation of the thermal and mechanical properties shows that phase separation between the two ‘polymers’ stabilizes the association polymer. The supramolecular ordered structure is characterised by the observation of a melting endotherm and birefringent textures in the material. The mechanical properties are comparable to thermoplastic elastomers based on covalent multiblock copolymers with high modulus and tensile strength.     

Elastin covalent structure as determined by solid phase amino acid sequencing

The amino acid sequences of 16 large tryptic fragments of aortic tropoelastin have been determined establishing the presence of several repeating structures: GVP, GGVP, PGVGV, PGVGVA, and AGVPGFGVG. The methodologies for achieving these results by solid phase sequencing are reviewed and also the possible biologic significance of the unusual primary structures of elastin are discussed.     

HIO4-oxidized soluble polysaccharides as polyfunctional links for covalent binding of enzymes, 1. Preparation of polysaccharides and matrices for their binding

Soluble hydrophillc polyaldehydes can be used as linking and crosslinking reagents for the modification and immobilization of proteins. An imine bond (or possibly an α-hydroxyamino bond) between NH₂ functions of proteins or matrices and the aldehyde functions of the soluble polymer is formed under mild conditions. The polyaldehydes used were HIO₄-oxidized soluble polysaccharides of different degrees of oxidation, and the NH₂ matrices were Enzacryl AA and Enzacryl AH (for reference purposes also aminohexylcellulose), macroporous copolymer of glycidyl methacrylate reacted with ammonia, and nylon 6 and Estapor latices both with surface amino groups. Dextrans and Zulkowski starch were readily oxidized with HIO₄, the degree of oxidation being determined by means of a modified photometric aldehyde determination method. As regards the reaction of the matrices with the polyoxidized polysaccharides (“glycosidation of the matrix”), it was found that 20 h incubation at 20°C and phosphate buffer pH = 6 furnished good results. In the investigations discussed in the present paper, relatively large quantities of polysaccharide were used for incubation, in order to achieve a high degree of glycosidation. For a closer analysis of matrices, several methods for the determination of the NH₂ concentrations on matrices were examined; in this case the reaction with pentafluorobenzaldehyde in aqueous phase followed by fluoride-analysis furnished well reproducible results.