Methacrylated glycol chitosan as a photopolymerizable biomaterial

Glycol chitosan is a derivative of chitosan that is soluble at neutral pH and possesses potentially useful biological properties. With the goal of obtaining biocompatible hydrogels for use as tissue engineering scaffolds or drug delivery depots, glycol chitosan was converted to a photopolymerizable prepolymer through graft methacrylation using glycidyl methacrylate in aqueous media at pH 9. N-Methacrylation was verified by both (1)H NMR and (13)C NMR. The degree of N-methacrylation, measured via (1)H NMR, was easily varied from 1.5% to approximately 25% by varying the molar ratio of glycidyl methacrylate to glycol chitosan and the reaction time. Using a chondrocyte cell line, the N-methacrylated glycol chitosan was found to be noncytotoxic up to a concentration of 1 mg/mL. The prepolymer was cross-linked in solution using UV light and Irgacure 2959 photoinitiator under various conditions to yield gels of low sol content ( approximately 5%), high equilibrium water content (85-95%), and thicknesses of up to 6 mm. Cross-polarization magic-angle spinning (13)C solid state NMR verified the complete conversion of the double bonds in the gel. Chondrocytes seeded directly onto the gel surface, populated the entirety of the gel and remained viable for up to one week. The hydrogels degraded slowly in vitro in the presence of lysozyme at a rate that increased as the cross-link density of the gels decreased.     

Development of advanced antimicrobial and sterilized plasma polypropylene grafted muga (antheraea assama) silk as suture biomaterial

Surface modification of silk fibroin (SF) materials using environmentally friendly and non‐hazardous process to tailor them for specific application as biomaterials has drawn a great deal of interest in the field of biomedical research. To further explore this area of research, in this report, polypropylene (PP) grafted muga (Antheraea assama) SF (PP‐AASF) suture is developed using plasma treatment and plasma graft polymerization process. For this purpose, AASF is first sterilized in argon (Ar) plasma treatment followed by grafting PP onto its surface. AASF is a non‐mulberry variety having superior qualities to mulberry SF and is still unexplored in the context of suture biomaterial. AASF, Ar plasma treated AASF (AASF_Ar) and PP‐AASF are subjected to various characterization techniques for better comparison and the results are attempted to correlate with their observed properties. Excellent mechanical strength, hydrophobicity, antibacterial behavior, and remarkable wound healing activity of PP‐AASF over AASF and AASF_Ar make it a promising candidate for application as sterilized suture biomaterial. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 355–365, 2014.     

Chitosan gallate as potential antioxidant biomaterial

Chitosan gallate were synthesized using a free radical-induced grafting reaction. Chitosan gallate showed enhanced water-solubility compared to plain chitosan, and exhibited good thermal stability. The IC₅₀ value of chitosan gallate against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was 17.86 μg/mL. In addition, chitosan gallate effectively inhibited the generation of intracellular reactive oxygen species (ROS), and also suppressed lipid peroxidation in RAW264.7 macrophage cells. Chitosan gallate also exhibited the protection effect on genomic DNA damage by induced hydroxyl radical, and up-regulated the protein expression of antioxidant enzymes including superoxide dismutase-1 and glutathione reductase under H₂O₂-mediated oxidative stress in RAW264.7 macrophage cells. These results indicate that chitosan gallate might be potential antioxidant biomaterials.     

Fish skin as a biomaterial for halal collagen and gelatin

Around 40% of the total catch weight of fish is regarded as byproducts, consisting of skin, fins, bones, scales, viscera, etc. The utilization of these byproducts is important to increase their commercial values as well as to prevent environmental pollution. Meanwhile, nowadays, it is getting a global trend to provide foods and other industrial materials which have been accredited as halal products for Moslem communities. As a way of processing fish byproducts to meet the halal criteria, preparation of collagen and gelatin would be useful to fulfill the market demand. As a result of screening studies on fishery byproducts, fish skin has been found to be the good source for halal collagen and gelatin, which show satisfactory quality compared with those from bovine sources which could cause bovine spongiform encephalopathy (BSE).     

Collagen/hyaluronan membrane as a scaffold for chondrocytes cultivation

Rabbit chondrocytes were cultivated in vitro using the collagen/hyaluronan membrane. The membrane did not show any adverse effects on chondrocyte viability during in vitro cultivation. The inoculated cells grew without any negative changes. According to the histochemical analyses: (i) hematoxylin and eosin; (ii) safranin O; and (iii) rabbit anti-human collagen type II staining, the rabbit chondrocytes maintained their morphology and phenotype during in vitro cultivation. The collagen/hyaluronan membrane became more stable and stiffer after long time cultivation. The proliferation of the chondrocytes stabilised the structure of the membrane. The collagen/hyaluronan membrane is suitable material for the chondrocyte growth and could provide functional tissue-engineered scaffold for cartilage repair.     

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

Development of proteomic tools to study protein adsorption on a biomaterial, titanium grafted with poly(sodium styrene sulfonate)

It is known that protein adsorption is the initial interaction between implanted biomaterials and biological environment. Generally, a complex protein layer will be formed on material surfaces within a few minutes and the composition of this layer at the interface determines the biological response to the implanted material, and therefore the long-term compatibility of the biomaterial. Despite different techniques exist to observe protein adsorption on biomaterials, none of them led to the identification of adsorbed proteins. In this paper, we report a chromatographic technique coupled to proteomics to analyse and identify proteins from complex biological samples adsorbed on biomaterial surfaces. This approach is based on (1) elaboration of the chromatographic support containing the biomaterial (2) a chromatography step involving adsorption of proteins on the biomaterial (3) the high-resolution separation of eluted proteins by 2-DE gel and (4) the identification of proteins by mass spectrometry. Experiments were performed with proteins from platelets rich plasma (PRP) adsorbed on a biomaterial which consist in titanium bioactivated with PolyNaSS. Our results show that chromatographic approach combined to 2-DE gels and mass spectrometry provides a powerful tool for the analysis and identification of proteins adsorbed on various surfaces.     

Association of a synthetic bone graft and bone marrow cells as a composite biomaterial

Porous calcium phosphates have osteoconductive properties. The aim of this study was to obtain synthetic calcium phosphate bone graft substitute. X-ray diffraction was employed to investigate the formation of the beta-tricalcium phosphate (β-TCP) phase. We evaluated the effects of bone marrow on the osteoconductivity and mechanical properties of synthetic bone graft (SG). SG cylinders loaded with bone marrow (SGBM) and SG alone were implanted into rabbits femoral condyle bone defects. Histological examinations revealed the resorption of the SG, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous β-TCP by newly formed bone. Histomorphometry conducted after three months revealed the osteoid surface to be higher in SGBM than SG (p < 0.05). The compressive strengths of SG and SGBM were significantly higher than the anatomic control at all time periods. The elastic modulus of SBG and SGBM became weaker after implantation. The present results indicate that gB-TCP is a good matrix for bone marrow, which contributes osteoinductive properties in an orthotopic. The composite biomaterial may be useful in reconstructive bone surgery.     

Silk as an innovative biomaterial for cancer therapy

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.     

Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.     

Chalcone embedded polyurethanes as a biomaterial: Synthesis, characterization and antibacterial adhesion

An antibacterial dimethylamino-chalcone embedded multiblock copolymer (PCL-PEG) was synthesized and characterized using FT-IR, ¹H NMR, SEM and SEC and the compound was characterized using FT-IR, ¹H NMR, and ¹³C NMR. A 10% copolymer composite was prepared and casted as film to be used as a biomaterial and the copolymer films without the compound acted as control. TGA, DSC, AFM, SEM and EDAX analysis were performed for the above samples. Surface roughness (R_a) of the copolymer composite film was less when compared to the copolymer film which indicated the proper distribution of chalcone in the composite film. copolymer composite film was hydrophilic compared to copolymer film. Antibacterial adhesion studies were performed for copolymer composite polymer film and evaluated using CFU measurement and SEM analysis. Copolymer composite film shows promising antibacterial adhesion compared to the copolymer film. Hence the copolymer composite film can be used as a new biomaterial endowed with antibacterial properties.     

Chitosan-vancomysin composite biomaterial as a laser activated surgical adhesive with regional antimicrobial activity

We have used laser irradiation to enhance the natural adhesiveness of chitosan to form a thin film surgical adhesive. Prevention of infection at surgical sites often utilizes systemic provision of antibiotics with reduced local efficacy and potential side effects. In the work reported here, we investigate the bactericidal properties of laser-irradiated chitosan films and their impregnation with the antibiotic vancomycin. Despite strong efficacy in solution, chitosan films showed no antimicrobial activity against representatives of common pathogens Escherichia coli , Staphylococcus aureus , and S. epidermidis . In contrast, a composite of chitosan adhesive and the antibiotic vancomycin showed therapeutically significant release profiles greater that the Minimum Bactericidal Concentrations (MBCs) for the Staphylococci over a 28 day period. These composite films had greater crystallinity, up to 28 ± 3 compared to 8.9 ± 2%, for its unblended counterpart. Despite a significant increase in material strength from 31.4 ± 4 to 77.5 ± 5 MPa, flexibility was still maintained with an elongation to break around 5 ± 2% and fold endurance of approximately 30 ± 3-folds. Laser irradiation had no apparent effect on the release or activity of the antibiotic which survived transient temperatures at the film-tissue interface during infrared irradiation of around 54 °C. Furthermore, significant adhesive strength was still apparent, 15.6 ± 2 KPa. Thus, we have developed a laser-activated bioadhesive with the potential to close wounds while facilitating the prevention of microbial infection through local release of antibiotic targeted to the site of potential infection.     

Synthesis and physicochemical and dynamic mechanical properties of a water-soluble chitosan derivative as a biomaterial

The physicochemical and rheological properties of a water-soluble chitosan (WSC) derivative were characterized in order to facilitate its use as a novel material for biomedical applications. The WSC was prepared by conjugating glycidyltrimethylammonium chloride (GTMAC) onto chitosan chains. Varying the molar ratio of GTMAC to chitosan from 3:1 to 6:1 produced WSCs with a degree of substitution (DS) that ranged from 56% to 74%. The WSC with the highest DS was soluble in water up to concentrations of 25 g/dL at room temperature. An increase in the polymer concentration gradually increased both the pH and conductivity of the WSC solutions. The rheological properties of the WSC solutions were found to be dependent on the salt and polymer concentrations as well as the DS value. In the absence of salt, the rheological behavior of the WSC was found to be typical of that for a polyelectrolyte in the dilute solution regime. However, the addition of salt decreased the viscosity of the polymer solution due to the reduction of electrostatic repulsions by the positively charged trimethylated ammonium groups of the WSC. In the concentrated regime, the viscosity of the WSCs was found to follow a power-law expression. The lowest DS WSC had the more favorable viscoelastic properties that were attributed to its high molecular weight, as confirmed by the stress relaxation spectra and intrinsic viscosity measurements. The effect of DS on the degree of interaction between WSC and the lipid egg phosphatidylcholine was investigated by FTIR analysis. Overall, the lower DS WSC had enhanced rheological properties and was capable of engaging in stronger intermolecular physical interactions.     

Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.     

Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial

Mytilus galloprovincialis foot protein type-5 (Mgfp-5) is one of the mussel adhesive proteins that participate in adhesion with the substratum. We previously reported the production of recombinant Mgfp-5 in Escherichia coli and showed that the recombinant protein had superior adhesion abilities versus those of Cell-Tak, a commercially available mussel adhesive protein mixture. In the present work, we investigated the feasibility of using recombinant Mgfp-5 as a cell adhesion agent. Purified and tyrosinase-modified recombinant Mgfp-5 was used to adhere living anchorage-independent cells such as insect Drosophila S2 cells and human MOLT-4 cells onto glass slides. Our results revealed that these cell lines efficiently attached to recombinant Mgfp-5-coated glass surfaces, and that surface-immobilized S2 cells were viable and able to undergo cell division for up to 1 week. Cytochemical studies with 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei and immunofluorescence for secreted foreign human erythropoietin (hEPO) from recombinant S2 cells and quantitative comparative analyses of S2 cell binding ability with Cell-Tak and poly-L-lysine, the main cell adhesion agent, were performed to demonstrate successful usage of recombinant Mgfp-5 for cell biological applications. Collectively, these results indicate that recombinant Mgfp-5 may be a useful new cell adhesion biomaterial for anchorage-independent cells.     

Dietary protein as a potent regulator of the hyaluronan synthase gene in rat skin

The status of hyaluronan, the major glycosaminoglycan in the skin, is regulated by many factors such as cytokines and glucocorticoids. To examine whether and how protein malnutrition affects the status of skin hyaluronan, the hyaluronan content and mRNA levels of hyaluronan synthases (has) were analyzed in the skin of rats fed on a protein-free diet or on a 12% gluten diet. When these malnourishing diets had been given for 1 week, the hyaluronan content was significantly reduced as compared with that in rats fed on a 12% casein diet. Substantial falls in the mRNA levels of rhas2 and rhas3 were also observed. The reduction of mRNAs was already evident on the second day of treatment with the malnourishing diets. These results suggest that protein malnutrition has a primary impact on the gene expression of rhass, which leads to the reduction of hyaluronan content and to disfunction of the skin.     

Care during freeze-drying of bovine pericardium tissue to be used as a biomaterial: a comparative study

Bovine pericardium (BP) tissue is widely used in the manufacture of bioprosthetics. The effects of freeze-drying on the BP tissue have been studied by some researchers in order to decrease their cytotoxicity due to preservation in formaldehyde solution, and to increase the lifetime of the product in storage. This study was undertaken in order to study the effect of freeze-drying in the structure of BP. To perform this study BP samples were freeze-dried in two different types of freeze-dryers available in our laboratory: a laboratory freeze-dryer, in which it was not possible to control parameters and a pilot freeze-dryer, wherein all parameters during freezing and drying were controlled. After freeze-drying processes, samples were analyzed by SEM, Raman spectroscopy, tensile strength, water uptake tests and TEM. In summary, it has been demonstrated that damages occur in collagen fibers by the loss of bulk water of collagen structure implicating in a drastic decreasing of BP mechanical properties due to its structural alterations. Moreover, it was proven that the collagen fibrils suffered breakage at some points, which can be attributed to the uncontrolled parameters during drying.     

Pep-1 as a novel probe for the in situ detection of hyaluronan.

Hyaluronan (HA) is expressed by most tissues, including skin. Localization of HA in the skin is assessed by histology with HA-binding protein (HABP) serving as the probe. Reports have suggested that HA expression in skin is altered in a number of diseases. However, interlaboratory variations in HABP staining profiles, even in normal skin, suggest a need to standardize methods and/or identify new probes. We report the staining patterns of a HA-binding peptide (termed "Pep-1") in human and mouse skin. After acetone fixation, Pep-1 stained HA in the intercellular spaces of the epidermis, whereas staining in the dermis was weak and diffuse in both human and mouse skin. HABP staining of the epidermis and dermis were comparable in human skin but failed to stain the vital epidermis of mouse skin. In human skin, Pep-1 stained the basal, spinous, and granular layers, whereas HABP failed to stain the basal layer. Precipitation of HA in situ resulted in dermal staining but weak staining in the epidermis for HABP and Pep-1. Our results may suggest that Pep-1 is sensitive to HA conformation. Furthermore, Pep-1 may represent a new probe to study HA expression in the skin.     

Microwave initiated synthesis of polyacrylamide grafted Psyllium and its application as a flocculant

This paper reports a novel microwave initiated method for synthesis of polyacrylamide grafted Psyllium (Psy-g-PAM). Psyllium was modified through grafting of polyacrylamide (PAM) chains on it using microwave radiations only, in absence of any other free radical initiator. The grafting was confirmed by intrinsic viscosity study and characterization techniques like FTIR spectroscopy, elemental analysis (C, H, N, O and S) and SEM morphology study. Further, the flocculation efficacy of the synthesized graft copolymers was studied in kaolin and coal fine suspension through standard 'Jar test' procedure.