Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.     

Studies on chitin, 3. Preparation of pure chitin,poly(N-acetyl-D-glucosamine), from the water-soluble chitin

Pure chitin, poly(N-acetyl-D -glucosamine), was successfully prepared by acetylating the partially deacetylated water-soluble chitin with acetic anhydride-pyridine or with acetic acid-dicyclohexylcarbodiimide. Although enough selectivity between amino and hydroxyl groups was not shown, acetylation with acetic anhydride-pyridine gave rise to the complete acetylation of amino groups and thereby O-, N-acetylated chitin was formed. The ester groups were then selectively transformed to hydroxyl groups by means of either hydrolysis or transesterification to give the pure chitin. Acetic acid-dicyclohexylcarbodiimide system was found to enable one step complete and selective acetylation of amino groups under very mild conditions and it appeared to be superior to the others for the purpose.     

High molecular weight chitosan 6-O-sulfate. Synthesis,ESR and NMR characterization

Selective sulfation of the primary hydroxyl group of chitosan was performed by means of the pyridine-SO₃ complex after the positions 2 and 3 of the aminoglucopyranosidic ring were protected by coordination with copper ions. The requirements for complexing copper ions were investigated as a function of the pH and Cu(II)/chitosan mole ratio of the starting solution. Cu(II)-chitosan complexes were characterized by ESR and ¹³C NMR CP-MAS investigations; two different paramagnetic Cu(II) species were observed, whose relative quantity changes by changing the pH of the starting solution. The g tensor values (species I: g_1‖ = 2,28, g_1‖ = 2,06, A_2‖ = 160 G; species II; g_2‖ = 2,24, g_2‖ = 2,06, A_2‖ = 160 G) are consistent with a CuN₂O₂ chromophore. The sulfation reaction was studied in the range of 25 to 75°C using 2 : 1 to 6 : 1 pyridine-SO₃/Cu(II)-chitosan mole ratios. Under optimized reaction conditions, a selective 6-O sulfation was obtained in more than 90% yield.     

Surface characterization and platelet compatibility evaluation of surface-sulfonated chitosan membrane

The effect of various sulfonated derivatives of chitosan on platelet activation and blood coagulation was examined. The surface properties of artificial biomaterials have been thought as the key factors which mediate the interactions between the biological environment and biomaterial itself. In this study, the sulfonation was directly performed on the chitosan membrane surface. The chitosan membrane was surface-sulfonated by reactions with sulfur-pyridine trioxide complex (SO₃/pyridine) in H₂O solution and N,N-sulfur-dimethylformamide trioxide complex (SO₃/DMF) in DMF.Blood compatibility was evaluated by an in vitro platelet adhesion assay. The surface reaction of SO₃/pyridine in aqueous acid medium yields N,O-sulfated chitosan with cationic NH⁺ ₃ groups. After neutralization, this surface has been shown to induce a low degree of platelet adhesion and activation. When the surface-sulfonation is performed in an aqueous alkaline medium, although the degree of sulfonation is lower than the samples above, the N-sulfated chitosan significantly reduced the adhesion and activation of platelets. For the acidic SO₃/DMF reaction system, N,O-sulfated chitosan was obtained with a high extent of sulfonation and cationic NH3+groups. On this surface fully spread platelets and some platelet aggregates were found instead. This may be attributed to the ionic interactions between the platelets membrane surface and the cationic groups on the modified chitosan membrane.     

Straightforward Determination of the Degree of N‐Acetylation of Chitosan by Means of First‐Derivative UV Spectrophotometry

First‐derivative UV spectrophotometry is shown to be a reliable method for the determination of the degree of N‐acetylation of chitosan samples. A mathematical expression is derived that allows to determine the DA directly from the mass concentration of a chitosan solution and the first derivative of its UV spectrum at 202 nm, thus eliminating the need for empiric correction curves for highly deacetylated samples. A procedure is proposed for the accurate mass determination of the hygroscopic chitosan. The proposed approach facilitates the routine determination of the DA, especially when using potent multiwell microplate readers, which allow hundreds of samples to be measured in just a few minutes.

     

Trägerkatalysatoren aus C12-allylnickel(II)-komplexen [Ni(C12H19)]X (X = SbF6, O3SCF3) und amorphem aluminiumfluorid in toluol als modell-katalysatoren für das technische katalysatorsystem Ni(octanoat)2/BF3·OEt2/AlEt3 zur 1,4-cis-polymerisation des butadiens

The η³, η², η²-dodeca-2(E), 6(E), 10(Z)-trien-1-yl-nickel(II) complexes [Ni(C₁₂H₁₉)]X (X = SbF₆, O₃SCF₃) were treated in toluene with amorphous aluminium trifluoride (which was prepared from AlEt₃ and BF₃ · OEt₂) in a mole ratio 1 : 10 to 20, forming a highly active catalyst for the 1,4-cis polymerization of butadiene. This catalyst is comparable in its activity and selectivity, and in the molar mass distribution of the polybutadiene, with the technical nickel catalyst Ni(O₂CR)₂/BF₃?OEt₂/AlE₃ developed by Bridgestone Tire Company thirty years ago. The existence of the C₁₂-allynickel(II) cation [Ni(C₁₂H₁₉)]⁺ on the AlF₃ support could be proved by FAB mass spectroscopic measurements. In agreement with our reaction model for the allyl nickel complex catalyzed butadiene polymerization, it is concluded that the technical nickel catalyst in its effective structure can be described as a polybutadienylnickel(II) complex co-ordinated to a polymeric fluoroaluminate anion via a fluoride bridge.     

Tetronic-grafted chitosan hydrogel as an injectable and biocompatible scaffold for biomedical applications

In recent years, injectable chitosan-based hydrogels have been widely studied towards biomedical applications because of their potential performance in drug/cell delivery and tissue regeneration. In this study, we introduce a simple and organic solvent-free method to prepare tyramine–tetronic–grafted chitosan (TTeC) via activation of four terminal hydroxyl groups of tetronic, partial tyramine conjugate into the activated product and grafting the remaining activated moiety of tetronic-tyramine onto chitosan. The grafted copolymer was well characterized by UV–Visible, ¹H NMR, and Thermogravimetric analysis. The aqueous TTeC copolymer solution rapidly formed hydrogel in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂) at physiological conditions. The gelation time of the hydrogel was performed within a time period of 4–60?s, when the concentrations of HRP, H₂O₂, and polymers varied. The hydrogel exhibited highly porous structure which could be controlled by using H₂O₂. In vitro cytotoxicity study with Human Foreskin Fibroblast cell using live/dead assay indicated that the hydrogel had high cytocompatibility and could play a role as a scaffold for cell adhesion. The injectable hydrogels did not cause any inflammation after two weeks and one day of the in vivo injection. The obtained results demonstrated a great potential of the TTeC hydrogel in biomedical applications.     

Polymerization and asymmetric oligomerization of allylsilanes using chiral ethylenebis(4,5,6,7-tetrahydro-1-indenyl)zirconium and -hafnium complexes

Stereospecific polymerization and asymmetric oligomerization of allylsilanes were investigated by using C_2- and C_s- symmetric zirconocene catalysts. Isotactic and syndiotactic poly(allylsilane)s were produced with rac-ethylenebis(4,5,6,7-tetrahydro-1-indenyl)zirconium dichloride ( 1 ) and diphenylmethylene(η⁵-cyclopentadienyl)(η⁵-fluorenyl)zirconium dichloride ( 6 ), respectively. Bulky allylsilanes afforded optically active oligomers in the reactions using optically active ethylenebis(4,5,6,7-tetrahydro-1-indenyl)zirconium and -hafnium complexes.     

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.     

New polymer syntheses, 3. Binding of nucleosides to basic polypeptides via isocyanato-isothiocyanates

New aliphatic α-isocyanato-ω-isothiocyanates of various chain lengths were synthesized from ω-isothiocyanatocarboxylic acid chlorides and trimethylsilyl azide. The 5′-OH group of 2′-O,3′-O-isopropylideneuridine and of N⁶-benzoyl-2′-O,3′-O-isopropylideneadenosine was added selectively onto the isocyanate group of aromatic and aliphatic α-isocyanato-ω-isothiocyanates. The isothiocyanate group of the resulting nucleoside derivatives was then reacted with the free amino groups of polylysine, isopolylysine, polyornithine and isopolyornithine. The resulting polypeptides with pending nucleosides were characterized by elemental analyses and 360 MHz ¹H NMR spectra. A new “one-pot-procedure” was developed to protect the primary amino group (N⁶) of 2′-O,3′-O-isopropylideneadenosine.     

Comparison of atriopeptins II and III,VIP and β2--adrenoceptor-evoked relaxations of the two layers of smooth muscle in the rat portal vein

Comparison of atriopeptins II and III, VIP and β₂-_-adrenoceptor-evoked relaxations in the two layers of smooth muscle in the rat portal vein. Acta Physiol Stand 130, 593–599. Received II November 1986, Accepted 26 February 1987. ISSN 0001–6772. Department of Physiology, University of Bergen, Norway. The relative importance of vascular relaxations induced by atriopeptins (AP), the β-adrenoceptor agonist isoprenaline and of the neuropeptide VIP was studied in uitro on circular and longitudinal preparations of the rat portal vein. Two members of the rat atriopeptins (AP II and III) were equipotent with respect to relaxation of the spontaneously contracting outer, longitudinal layer and of the α₁-_-contracted inner, circular layer. The potency for AP II was about 13 times lower in the inner (pD₂= 7.48±0.73, n= 6) than in the outer layer (pD₂= 8.60 ±0.34, n = 6). No significant difference was apparent between the intrinsic activities for AP II in the two layers. The potencies for AP II were for both layers higher than those for VIP while the intrinsic activities for AP II were significantly lower than for VIP and for the reference agonist, isoprenaline in both layers. Atriopeptin II was equally efficient in relaxing the K⁺-depolarized and a₁-_-contracted longitudinal segments. Neither the β-antagonist, propranolol nor the guanylate cyclase inhibitor, methylene blue, modified the potency or the intrinsic activity of AP II. These results suggest that concentrations of circulating atriopeptins above 10 nM may contribute to reduction of vascular tone by the methylene blue insensitive receptors for AP II and III in the portal-mesenteric vein region.     

Poly(vinylsaccharide)s, 4. Synthesis and polymerization of 6-o-methylallylgalactose derivatives

Methylallyl ethers of 1,2:3,4-di-O-isopropylidene-D-galactopyranose and 1,2:3,4-di-O-cyclohexylidene-D-galactopyranose were synthesized as new monomers containing saccharide moieties and copolymerized with maleic anhydride. The hitherto unknown 6-O-(2-alkoxycarbonylallyl)-1,2:3,4-di-O-isopropylidene-D-galactopyranoses ( 8a and 8b; alkyl: methyl and ethyl, respectively) were prepared and polymerized under free radical conditions. The substances were characterized by ¹³C NMR, elemental analyses and molecular weight determinations. Water solubility of these polymers was obtained by removing the isopropylidene protecting groups. The inherent viscosities of these polymers, measured in 0,1 M Na₂SO₄ aqueous solution, were of the same order of magnitude as those of polyacrylamides of the corresponding degrees of polymerization.     

Handelsübliche polymere als träger in der oligonucleotidsynthese, 1. Synthese eines pentanucleosidtetraphosphats an merckogel or 1000 000®

Merckogel OR 1000000®, a commercial macroreticular polyvinylacetate resin, was partially saponified and loaded with nucleoside (0,5 mmol/g) by reaction with 3′-O-(β-benzoylpropionyl)-5′-thymidyl chloroformate. The synthesis of oligonucleotides on this carrier was done in three steps per chain extension, namely 1. the phosphorylation of the 3′-OH groups with β-cyanoethyl phosphate, 2. the condensation with 3′-O-(β-benzoylpropionyl)thymidine, and 3. cleavage of the benzoylpropionyl groups. Steps 1 and 3 occurred almost quantitatively and step 2 in ca. 50% yield. The reaction cycle was gone through four times and, thus, the pentanucleoside tetraphosphate dT (pT)₄ ( 11 ) was synthesized in 6% overall yield. A device for the micromanipulation of support resins is described.     

Attachment,Spreading, and Adhesion Strength of Human Bone Marrow Cells on Chitosan

The successful integration of an orthopedic implant into bone depends on the mechanisms at the tissue–implant interface and mostly on the osteoblast attachment phenomenon. Chitosan has emerged as an attractive biomacromolecule favoring osseointegration. In this study highly deacetylated chitosan coatings, with roughness of about 1 nm, were bonded to glass surfaces via silane–glutaraldehyde molecules. Human osteoblasts were used to study the development of attachment during the first 60 min. Chitosan favored the number of the attached cells compared to the uncoated surfaces for 30 min seeding time (t _s). For t _s up to 60 min the attached cell area was almost 210% significantly higher on the chitosan surfaces, indicating an enhanced spreading process. To determine the cell attachment strength, a micropipette aspiration method was used, where the value of the term I = ∫Fdt is representative of the single cell attachment–adhesion procedure and quantitatively reflects the strength evolution during attachment: F equals the detaching force applied on the cell. The results showed higher strength values on the chitosan surfaces. The findings reinforce the favorable environment of the biomacromolecule for the osteoblast and the new approach regarding the quantitatively evaluation of adhesion provides important contribution for the study of cell–material interaction, especially during the crucial first phase of cell attachment.     

Synthesis of an acryloyl-type polymer pending D-glucose analog of N-acetylmuramyl-L-alanyl-D-isoglutamine

Poly{1-{3-[O-1-(1-carboxyethylaminocarbonyl)ethyl]-6-O-D -glucopyranosylcarbonyl}ethylene} (3b) was synthesized as the lipophilic and polymeric model of 1-{N-[2-(2-acetylamino-2-deoxy-3-O-D -glucopyranosyl)propionyl]-L -alanylamino}glutamic acid (1) to the minimum required structure for the eimmunoadjuvant activity of bacterial cell wall. N-[2-(6-O-acryloyl-1,2-O-isopropylidene-α-D -3-O-glucofuranosyl)propionyl]-L -alanine benzyl ester (6) was prepared as a key monomer in the synthesis. The homopolymerization of this monomer was carried out in benzene at 50°C using 2, 2′-azoisobutyronitrile as radical initiator to give polymer 7 . From the results of copolymerization with styrene, the values of Q and e for the acryloyl type monomer 6 were estimated to be 2,0 and 0,11, respectively. The removal of isopropylidene and benzyl protecting groups from homopolymer 7 was carried out by acidic treatment with trifluoroacetic acid/water (vol. ratio 6:1) and by catalytic hydrogenolysis with palladium carbon in acetone, respectively, to afford the homopolymer 3b .     

Protein adsorption on preadsorbed polyampholytic monolayers

The adsorption behaviour of five different globular proteins on pure silicon substrates and on preadsorbed polyampholytic monolayers has been investigated as a function of protein concentration. The prelayers were prepared by adsorption of the ampholytic diblock copolymer poly(methacrylic acid)-block-poly((dimethylamino)ethyl methacrylate)(PMAA-b-PDMAEMA). This polyampholyte adsorbs in densely packed micelles directly from aqueous solution. Ellipsometry was used to determine the amount of adsorbed polyampholyte and protein. While ATR-IR spectroscopy gives information about the adsorption and desorption behaviour of the preadsorbed polyampholytic layer, the lateral structures of the dried films were investigated by scanning force microscopy (SFM). The amount of protein adsorbed was found to be strongly influenced by the preadsorbed polyampholyte compared to the adsorption on the pure silicon substrates. No displacement of the polyampholyte by the proteins was detected. In most cases the protein adsorption was reduced by the preadsorbed polyampholytic layer. The observed trends are explained by the change in electrostatic and hydrophilic characteristics of the substrates. Furthermore, the entropy of adsorption has to be taken into account.     

Ischaemia-reperfusion induced alterations of mitochondrial function in hypertrophied rat heart

The impact of in vivo ischaemia and ischaemia-reperfusion (I-R) on mitochondrial respiratory function was investigated in hypertrophied (HP) hearts with aortic constriction compared with control hearts using an open-chest rat surgical model. Moreover, mitochondrial susceptibility to superoxide radicals (O₂P^-) in vitro was examined in HP and control hearts with or without I-R. With the site I substrates pyruvate-malate, mitochondrial state 4 (basal) respiration and the respiratory control index (RCI) were not affected by either ischaemia alone or I-R in both HP and control hearts. State 3 (ADP-stimulated) respiration was increased with I-R in control hearts, but showed a reduction after I-R in the HP hearts. Exposure of mitochondria to O₂P^- (20 nm hypoxanthine in the presence of 0.13 unit mL^-1 xanthine oxidase) significantly increased state 4 respiration, whereas state 3 respiration and RCI were decreased in all treatment groups. I-R hearts in both HP and control showed greater increases in state 4 respiration with O₂P^- than either sham or ischaemic hearts. HP hearts exhibited a significantly lesser extent of inhibition in state 3 respiration and RCI by O₂P^- compared with control hearts. These changes in mitochondrial respiratory properties were not observed with the site II substrate succinate. Myocardial reduced vs. oxidized glutathione ratio was significantly decreased after I-R in both control and HP hearts. Malondialdehyde content showed an increase with I-R, but the increase was significant only in control hearts. These data indicate that short-term in vivo I-R does not impair heart mitochondrial respiratory function, but renders the organelles more vulnerable to imposed oxidative stress. Mitochondria from the HP hearts are more resistant to free radical damage under normal and ischaemic conditions; however, this advantage is severely compromised after reperfusion.     

Untersuchungen zum oxidativen Abbau nativer Cellulose, 2. Einfluß der Alkalibehandlung auf die Viskositäts-Molekulargewichts-Beziehungen

For kinetic studies of cellulose degradation by molecular oxygen it is necessary to establish new [η]-M-relationships, since the treatment of cellulose by concentrated alkali solutions changes the intrinsic viscosity [η] without changing the molecular weight M. Two series of degradated celluloses treated for different times with sodium hydroxide solutions were prepared, and the constants of the Kuhn-Mark-Houwink equation were determined for cellulose nitrates in acetone and for celluloses in Cuoxam (Cu(NH₃)₄(OH)₂) by comparison of intrinsic viscosity with light scattering data.     

Chiral interaction of chitosan with azo dyes

Absorption and circular dichroism (CD) spectra were measured on complexes of highly deacetylated chitosans with azo dyes in 0,08% acetic acid. Blue and red shifts of the absorption peaks in the visible region were observed. The induced CD results suggest two different chiral configurations of the transition dipole moments of the dyes. Since the random sequence of amino and acetamido groups in deacetylated chitosan does not affect the induced CD per amino side-chain, intermolecular interactions of the dye molecules are most probable in these chitosan/dye systems.     

Soluble and film-forming cellulose derivatives with redox- chromogenic and enzyme immobilizing 1,4-phenylenediamine groups

Cellulose architectures with redox-chromogenic properties and anchor groups for the immobilization of biomolecules have been prepared by reaction of p-toluenesulfonic acid esters of cellulose (tosylcellulose, DS 2.3) with 1,4-phenylenediamine (PDA) in dimethyl sulfoxide (DMSO) solution at 100°C. The degree of substitution (DS) of the PDA groups and remaining p-toluenesulfonate (tosylate) groups, and thus the properties of the 6-deoxy-6-(4-aminophenyl)amino-2(3)-O-tosylcellulose (PDA cellulose) formed, can be adjusted by varying the PDA molar equivalents and the reaction time. Most of PDA celluloses are soluble in DMSO and N,N-dimethylacetamide (DMA) and form transparent films. The redox-chromogenic properties of PDA cellulose have been demonstrated by oxidation of the PDA groups with H₂O₂ to the corresponding diimine (λ_max = 480 nm) and by oxidative coupling with phenol to the corresponding indophenol (λ_max = 595 nm). The suitability of the PDA units as reactive anchor groups was shown by the immobilization of enzymes like glucose oxidase, peroxidase, and lactate oxidase. The highest enzyme activity achieved for peroxidase immobilized with glutardialdehyde on a PDA cellulose film was 0.165 U/cm².