Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications

Chitosan, a deacetylated derivative of chitin is a commonly studied biomaterial for tissue-engineering applications due to its biocompatibility, biodegradability, low toxicity, antibacterial activity, wound healing ability and haemostatic properties. However, chitosan has poor mechanical strength due to which its applications in orthopedics are limited. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has mechanical strength and osteoconductive property. In this work, HAp was deposited on the surface of chitosan hydrogel membranes by a wet chemical synthesis method by alternatively soaking the membranes in CaCl₂ (pH 7.4) and Na₂HPO₄ solutions for different time intervals. These chitosan hydrogel–HAp membranes were characterized using SEM, AFM, EDS, FT-IR and XRD analyses. MTT assay was done to evaluate the biocompatibility of these membranes using MG-63 osteosarcoma cells. The biocompatibility studies suggest that chitosan hydrogel–HAp composite membranes can be useful for tissue-engineering applications.     

Entrapment of β-galactosidase in polyvinylalcohol hydrogel

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g⁻¹) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.     

β-Cyclodextrin grafted polyethyleneimine hydrogel immobilizing hydrophobically modified glucose oxidase

Hydrogels which release their contents in response to glucose concentration were prepared by immobilizing glucose oxidase (GOD) into β-cyclodextrin grafted polyethyleneimine hydrogels (PEI-βCD hydrogel). For the tight immobilization, hydrophobically modified GOD (HmGOD) was prepared by reacting GOD with palmitic acid-N-hydroxysuccinimide ester (PA-NHS) in the molar ratio of 1:40. According to trinitrobenzene sulfonic acid (TNBS) assay, five palmitic acids were covalently attached to one GOD molecule. The activity of HmGOD was about 76% of native enzyme. The swelling ratios of HmGOD loaded hydrogels increased from about 960% to 1190% in 24h, when glucose concentration was varied from 0 to 100mg/dl. The % release in 48 h of fluorescein isothiocyanate dextran increased from about 53% to 89%, when glucose concentration was varied in the same range. Gluconic acid, produced by the enzymatic reaction, would protonate and swell the PEI-βCD hydrogel, leading to a higher release.     

Semicontinual synthesis of alkyl galactosides using β-galactosidase entrapped in polyvinylalcohol hydrogel

Fungal β-galactosidase from Aspergillus oryzae was immobilized into polyvinylalcohol (PVA) hydrogel by LentiKats® technology and used for the production of short-chain alkyl glycosides. Ethyl- and propyl-β-d-galactopyranosides were prepared from lactose (100?g/L) and varying initial amounts of alcohol (10–30% v/v) at 40?°C and pH 4.5. The entrapped β-galactosidase preserved 50% of the initial transgalactosylation activity after 25 repeated cycles in the production of ethyl β-d-galactopyranoside. When 5% (v/v) propanol was used as an acceptor, the enzyme activity (30–32?U/g immobilized enzyme) remained constant for 25 repeated batch runs. These findings suggest that entrapped β-galactosidase into LentiKats® has a great potential to be one effective, reusable and easy producible biocatalyst for the production of alkyl glycosides in a large scale.     

Growth modulation of fibroblasts by chitosan-polyvinyl pyrrolidone hydrogel: Implications for wound management?

Wounds in adults and fetuses differ in their healing ability with respect to scar formation. In adults, wounds lacking the epidermis exhibit excess collagen production and scar formation. Fibroblasts synthesize and deposit a collagen rich extracellular matrix. The early migration and proliferation of fibroblasts in the wound area is implicated in wound scarring. We have synthesized a hydrogel from chitosan-polyvinyl pyrrolidone (PVP) and examined its effect on fibroblast growth modulation in vitro. The hydrogel was found to be hydrophilic as seen from its octane contact angle (141.2+/-0.37 degrees). The hydrogel was non-toxic and biocompatible with fibroblasts and epithelial cells as confirmed by the 3(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) as-say. It showed dual properties by supporting growth of epithelial cells (SiHa) and selectively inhibiting fibro-blast (NIH3T3) growth. Growth inhibition of fibroblasts resulted from their inability to attach on to the hydrogel. These findings are supported by image analysis, which revealed a significant difference (P<0.05) between the number of fibroblasts attached to the hydrogel in tissue culture as compared to tissue culture treated polystyrene (TCPS) controls. However, no significant difference was observed (P>0.05) in the number of epithelial (SiHa) cells attached on to the hydrogel as compared to the TCPS control. Although in vivo experiments are awaited, these findings point to the possible use of chitosan-PVP hydrogels in wound-management.     

Impact of magnetic nanofillers in the swelling and release properties of κ-carrageenan hydrogel nanocomposites

Natural polysaccharides such as κ-carrageenan are an important class of biomaterials for drug delivery. The incorporation of magnetic nanoparticles in polysaccharide hydrogels is currently being explored as a strategy to confer to the hydrogels novel functionalities valuable for specific bio-applications. Within this context, κ-carrageenan magnetic hydrogel nanocomposites have been prepared and the effect of magnetic (Fe₃O₄) nanofillers in the swelling of the hydrogels and in the release kinetics and mechanism of a model drug (methylene blue) has been investigated. In vitro release studies demonstrated the applicability of the composites in sustained drug release. The mechanism controlling the release seems to be determined by the strength of the gel network and the extent of gel swelling, both being affected by the incorporation of nanofillers. Furthermore, it was demonstrated that the release rate and profile could be tailored using variable Fe₃O₄ nanoparticles load. Thus, this seems to be a promising strategy for the development of drug delivery systems with tailored released behavior.     

Synthesis, characterization and cytocompatibility studies of α-chitin hydrogel/nano hydroxyapatite composite scaffolds

α-chitin hydrogel/nano hydroxyapatite (nHAp) composite scaffold have been synthesized by freeze-drying approach with nHAp and α-chitin hydrogel. The prepared nHAp and nanocomposite scaffolds were characterized using DLS, SEM, FT-IR, XRD and TGA studies. The porosity, swelling, degradation, protein adsorption and biomineralization (calcification) of the prepared nanocomposite scaffolds were evaluated. Cell viability, attachment and proliferation were investigated using MG 63, Vero, NIH 3T3 and nHDF cells to confirm that the nanocomposite scaffolds were cytocompatible and cells were found to attach and spread on the scaffolds. All the results suggested that these scaffolds can be used for bone and wound tissue engineering.     

Calculation of the Three-Dimensional Structures of Two Antigenic Sequences, Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro and Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro, of the Circumsporozoite Protein From a Malaria-Causing Plasmodium

Using the chain build-up procedure based on the program ECEPP, we have computed the lowest energy structures for two terminally blocked subsequences from the antigenic circumsporozoite protein of Plasmodium berghei, that is known to cause malaria in animals. The full antigenic sequence is an octapeptide proline-rich tandem repeat, (Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp)₂. We computed the structures for the first octapeptide plus one Pro from the second octapeptide, terminally blocked CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–NHCH₃ as well as the first octpeptide with an additional three Pro residues from the adjoining unit, i.e., CH₃CO–Pro–Pro–Pro–Pro–Asn–Pro–Asn–Asp–Pro–Pro–Pro–NHCH₃. We find that the first sequence adopts a number of different low energy structures, the most probable of which has a probability of occurrence of 56 %. Addition of two more Pro residues results in the adoption a single, unique lowest energy structure that has a probability of occurrence of over 95 % without solvation effects and 86 % when solvation effects are included in the calculations. We predict that this structure may be the one recognized as a major antigenic determinant.     

Antibacterial activity and biocompatibility of a chitosan-γ-poly(glutamic acid) polyelectrolyte complex hydrogel

In this study, we prepared a polyelectrolyte complex (PEC) hydrogel comprising chitosan as the cationic polyelectrolyte and γ-poly(glutamic acid) (γ-PGA) as the anionic polyelectrolyte. Fourier transform infrared spectroscopy revealed that ionic complex interactions existed in the chitosan-γ-PGA PEC hydrogels. The compressive modulus increased upon increasing the degree of complex formation in the chitosan-γ-PGA PEC hydrogel; the water uptake decreased upon increasing the degree of complex formation. At the same degree of complex formation, the compressive modulus was larger for the chitosan-dominated PEC hydrogels; the water uptake was larger for the γ-PGA-dominated ones. Scanning electron microscopy images revealed the existence of interconnected porous structures (pore size: 30-100 μm) in all of the chitosan-γ-PGA PEC hydrogels. The chitosan-γ-PGA PEC hydrogels also exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, in vitro cell culturing of 3T3 fibroblasts revealed that all the chitosan-γ-PGA PEC hydrogels were effective in promoting cell proliferation, especially the positively charged ones (chitosan-dominated). Therefore, the chitosan-γ-PGA polyelectrolyte hydrogel appears to have potential as a new material for biomedical applications.     

Bone defect repair in rat tibia by TGF-β1 and IGF-1 released from hydrogel scaffold

Bone repair is one of the major challenges facing reconstructive surgery. Bone regeneration is needed for the repair of large defects and fractures. The ability of TGF-β1 and IGF-1 incorporated into hydrogel scaffold to induce bone regeneration was evaluated in a rat tibia segmental defect model. External fixation was performed prior to the induction of the segmental bone defect in order to stabilize the defect site. Hydrogel scaffold containing either TGF-β, IGF-1, TGF-β + IGF-1, hydrogel containing saline or saline, were inserted in the defect. Calcified material was observed in the defects treated with TGF-β 2 weeks following the start of treatment. Bone defects treated with TGF-β, IGF-1 or TGF-β + IGF-1 revealed significant bone formation after 4 and 6 weeks when compared to the control specimens. X-ray images showed that solid bone was present at the defect site after 6 weeks of treatment with TGF-β or TGF-β + IGF-1. A less pronounced bone induction was observed in the control specimens and bones treated with IGF-1. Percent closure ratio of bone defects after 6 weeks were 40, 80, 89, and 97% for saline, hydrogel, IGF-1, TGF-β and IGF-1 + TGF-β groups, respectively. It is concluded that hydrogel scaffold can serve as a good osteoconductive matrix for growth factors, and that it provides a site for bone regeneration and enhances bone defect healing and could be used as alternative graft material. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nonorthogonal tight-binding model with H–C–N–O parameterisation

A parametric nonorthogonal tight-binding model (NTBM1) with the set of parameters for H–C–N–O systems is presented. This model compares well with widely used semi-empirical AM1 and PM3/PM7 models but contains less fitting parameters per atom. All NTBM1 parameters are derived based on a criterion of the best agreement between the calculated and experimental values of bond lengths, valence angles and binding energies for various H–C–N–O molecules. Results for more than 200 chemical compounds are reported. Parameters are currently available for hydrogen, carbon, nitrogen, oxygen atoms and corresponding interatomic interactions. The model has a good transferability and can be used for both relaxation of large molecular systems (e.g., high-molecular compounds or covalent cluster complexes) and long-timescale molecular dynamics simulation (e.g., modelling of thermal decomposition processes). The program package based on this model is available for download at no cost from http://ntbm.info.     

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP–A. In addition to CENP–A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP–T–W–S–X complex. However, it is unclear how the CENP–T–W–S–X complex associates with centromere chromatin. Here, we demonstrate that the CENP–T–W–S–X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP–T–W–S–X complex primarily binds to DNA as a (CENP–T–W–S–X)₂ structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP–T–W–S–X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP–T or CENP–W, but not CENP–S or CENP–X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP–T–W–S–X complex. In summary, our work reveals the structural features and properties of the CENP–T–W–S–X complex for its localization to centromeres.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

Metabolomic changes in Caenorhabditis elegans lifespan mutants as evident from GC–EI–MS and GC–APCI–TOF–MS profiling

Lifespan mutants of the nematode Caenorhabditis elegans are a much studied aging model, however, aging-related changes at the metabolome level remain largely unexplored. To identify metabolic features connected to mitochondrial dysfunction, a hallmark of aging and age-related disease, we analyzed a short-lived mitochondrial mutant (mev-1(kn1)), a long-lived mutant with enhanced cellular maintenance (ife-2(ok306)) and the novel double mutant ife-2(ok306);mev-1(kn1) which is normal-lived, possibly through attenuation of the metabolic mev-1 phenotype. Metabolomic analysis involved coupled gas chromatography–mass spectrometry with electron ionization (GC–EI–MS) and, in addition, recently introduced GC with soft atmospheric pressure chemical ionization coupled to time-of-flight mass spectrometry (GC–APCI–TOF–MS) to yield complementary mass spectrometric information for enhanced metabolite annotation. Multivariate analysis allowed distinction of mev-1 and ife-2 mutants from the wild type, while suggesting still another, distinct metabolic phenotype for the ife-2;mev-1 double mutant. In mev-1(kn1), disturbed energy metabolism was indicated by upset TCA cycle homeostasis, elevated glycolytic substrate and lactic acid levels as well as depletion of free amino acids pools. Surprisingly, these mitochondrially related changes were retained in the ife-2;mev-1 mutant, as were highly elevated levels of the dipeptide glycylproline indicative of increased collagen catabolism. However, the double mutant reverted mev-1(kn1) changes in uric acid and long-chain fatty alcohol metabolism, two pathways connected to the peroxisomal compartment. Our results are in line with recent evidence for a critical role of this organelle in aging and demonstrate the usefulness of non-targeted metabolomics approaches for detecting complex metabolic changes in the study of mitochondrial dysfunction.     

News & Notes: Nucleotide Sequence of a DNA Fragment from Buchnera aphidicola (Aphid Endosymbiont) Containing the Genes aspS–trxB–serS–serC–aroA–rpsA–himD–tpiA

Buchnera aphidicola is an endosymbiont of aphids. The nucleotide sequence of an 11.5-kilobase DNA fragment from this prokaryotic organism was determined. Eight open reading frames were found coding for putative proteins involved in protein synthesis, serine and aromatic amino acid biosynthesis, as well as thioredoxin and carbohydrate metabolism. These results indicate that B. aphidicola has many genetic properties of free-living bacteria. Received: 31 December 1996 / Accepted: 6 January 1997     

Characterization of immunoreactive acetyl–Ser–Asp–Lys–Pro in human plasma and urine by liquid chromatography–electrospray mass spectrometry

The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography–electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.