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Improved sensitivity and decreased sample size in a cytotoxicity test for biomaterials: a modified colony microassay using a microplate and crystal violet staining
Tsuchiya, T., Y. Ikarashi, et al. (1994), J Appl Biomater 5(4): 361-7.
Abstract: Modified Eagle's minimum essential medium supplemented with 5% fetal calf serum was highly sensitive to cytotoxicity and formed large control colonies in the V79 colony assay. A highly sensitive cytotoxicity test was developed using 96-well microtiter plates. Test chemicals or extracts of polyurethane materials containing the same chemicals were added 24 h after inoculation of cell suspensions. The cells were fixed and stained with crystal violet after additional culture for 6 days (V79 cells) or 10 days (Balb/3T3 cells). In terms of sensitivity and rapid quantitative measurement, this modified colony microassay, using a low cell density in 96-well microplates, was superior to various cytotoxicity tests such as colony, growth inhibition, cytolethality, and agar diffusion assays.

Improvement of absorption enhancing effects of n-dodecyl-beta-D-maltopyranoside by its colon-specific delivery using chitosan capsules
Fetih, G., S. Lindberg, et al. (2005), Int J Pharm 293(1-2): 127-35.
Abstract: In general, absorption enhancing effects of various absorption enhancers were greater in the large intestine than those in the small intestinal regions. Therefore, the effectiveness of absorption enhancers is expected to be remarkably observed, if these enhancers can be delivered to the large intestine with some poorly absorbable drugs after oral administration. In this study, therefore, we examined whether chitosan capsules were effective for the colon-specific delivery of a certain absorption enhancer and can improve the absorption enhancing action of the absorption enhancer after oral administration. 5(6)-Carboxyfluorescein (CF) was used as a model drug to investigate the site-dependent effectiveness of various absorption enhancers by an in situ closed loop method. Sodium glycocholate (NaGC), n-dodecyl-beta-d-maltopyranoside (LM), sodium salicylate (NaSal) and sodium caprate (NaCap) were used as models of absorption enhancers in this study. Overall, the absorption enhancing effects of these enhancers for intestinal absorption of CF were greater in the colon than those in the jejunum and the ileum. Especially, among these enhancers tested in this study, LM showed much greater absorption enhancing effect in the colon than in the jejunum and the ileum. Therefore, LM was selected as a model absorption enhancer to examine the effect of chitosan capsules on the absorption enhancing effect of LM. When CF and LM were orally administered to rats using chitosan capsules, the plasma concentration of CF was much higher than those in other dosage forms including solution and gelatin capsules. Therefore, chitosan capsules may be useful carriers for colon-specific delivery of LM, thereby increasing its absorption enhancing effect from the intestinal membranes.

Improvement of ACE inhibitory activity of chitooligosaccharides (COS) by carboxyl modification
Huang, R., E. Mendis, et al. (2005), Bioorg Med Chem 13(11): 3649-55.
Abstract: In the present research, chitooligosaccharides (COS) were carboxylated with -COCH(2)CH(2)COO(-) groups to obtain specific structural features similar to Captopril. Angiotensin I converting enzyme (ACE) inhibitory activity of carboxylated COS was studied and observed to enhance its activity with increased substitution degree. Further, Lineweaver-Burk plot analysis revealed that inhibition was competitive via obligatory binding site of the enzyme. This was accompanied with substitution of positively charged quarternized amino groups to COS with different substitution degrees, in which negative impact on ACE inhibition was observed.

Improvement of gene delivery mediated by mannosylated dendrimer/alpha-cyclodextrin conjugates
Wada, K., H. Arima, et al. (2005), J Control Release 104(2): 397-413.
Abstract: The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)) bearing mannose (Man-alpha-CDE conjugates) with the various degrees of substitution of the mannose moiety (DSM) as a novel non-viral vector in a variety of cells. Man-alpha-CDE conjugates (DSM 3.3 and 4.9) were found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugates (DSM 1.1 and 8.3) in various cells, which are independent of the expression of cell surface mannose receptors. Cellular association of pDNA complexes with dendrimer, alpha-CDE conjugate and Man-alpha-CDE conjugate (DSM 3.3) and their cytotoxic effects differed only very slightly. Surface plasmon resonance study demonstrated that the specific binding activity of Man-alpha-CDE conjugates to concanavalin A was not very strong. Much more conjugation of the mannose moiety to alpha-CDE conjugates provided unfavorable physicochemical properties of pDNA complexes for gene transfer, e.g. the low interaction with pDNA, the low enzymatic stability of pDNA and the lack of pDNA compaction. Man-alpha-CDE conjugate (DSM 3.3) provided gene transfer activity higher than dendrimer and alpha-CDE conjugate in kidney 12 h after intravenous injection in mice. These results suggest the potential use of Man-alpha-CDE conjugate (DSM 3.3) as a non-viral vector.

Improvement of surface bioactivity on titanium by water and hydrogen plasma immersion ion implantation
Xie, Y., X. Liu, et al. (2005), Biomaterials 26(31): 6129-35.
Abstract: We have investigated the surface bioactivity of titanium after water and hydrogen plasma immersion ion implantation. Plasma immersion ion implantation (PIII) excels in the surface treatment of components possessing a complicated shape such as medical implants. In addition, water and hydrogen PIII has been extensively studied as a method to fabricate silicon-on-insulator (SOI) substrates in the semiconductor industry and so it is relatively straightforward to transfer the technology to the biomedical field. In our investigation, water and hydrogen were plasma-implanted into titanium sequentially. Our objective is that water PIII introduces near-surface damages that trap hydrogen implanted in the subsequent step to improve the surface bioactivity while the desirable bulk properties of the materials are not compromised. Ti-OH functional groups can be detected on the (H(2)O+H(2))-implanted titanium surface by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy. After incubation in simulated body fluids (SBF) for cytocompatibililty evaluation in vitro, bone-like hydroxyapatite was found to precipitate on the (H(2)O+H(2)) implanted samples while no apatite was found on titanium samples plasma implanted with water or hydrogen alone. Human osteoblast cells were cultured on the (H(2)O+H(2))-implanted titanium surface and they exhibited good adhesion and growth. Our results suggest a practical means to improve the surface bioactivity and cytocompatibility of medical implants made of titanium.

Improvement of the inhibitory effect of xanthones on NO production by encapsulation in PLGA nanocapsules
Teixeira, M., F. Cerqueira, et al. (2005), J Drug Target 13(2): 129-35.
Abstract: For the first time the inhibitory effect of xanthone and 3-methoxyxanthone on nitric oxide (NO) production by IFN-gamma/LPS activated J774 macrophage cell line is reported. A remarkable improvement of this effect promoted by encapsulation of these compounds in nanocapsules of poly (DL-lactide-co-glycolide) (PLGA) is also demonstrated. A weak inhibitory effect of 3.6% on NO production by activated macrophages was observed for xanthone at the highest studied concentration (100 microM). This effect was slightly higher for 3-methoxyxanthone at the same concentration, producing a reduction of 16.5% on NO production. In contrast, equivalent concentrations of xanthone and 3-methoxyxanthone incorporated in nanocapsules produced a significant decrease on NO production of 91.8 and 80.0%, respectively. Empty nanocapsules also exhibited a slight NO inhibitory activity, which may be due to the presence of soybean lecithin in the composition of the nanosystems. The viability of the macrophages was not affected either by free or nanoencapsulated xanthones. Fluorescence microscopy analysis confirmed that a phagocytic process was involved in the macrophage uptake of xanthone- and 3-methoxyxanthone-loaded PLGA nanocapsules. Phagocytosis might be the main mechanism responsible for the enhancement of the intracellular delivery of both compounds and consequently for the improvement of their biological effect.

Improving arterial prosthesis neo-endothelialization: application of a proactive VEGF construct onto PTFE surfaces
Crombez, M., P. Chevallier, et al. (2005), Biomaterials 26(35): 7402-9.
Abstract: The formation of a confluent endothelium on expanded polytetrafluoroethylene (PTFE) vascular prostheses has never been observed. This lack of endothelialization is known to be one of the main reasons leading to the development of thromboses and/or intimal hyperplasia. In this context, several efforts were put forward to promote endothelial cell coverage on the internal surface of synthetic vascular prostheses. The goal of the present study was to immobilize the vascular endothelial growth factor (VEGF) onto Teflon PTFE surfaces to generate a proactive polymer construct favoring interaction with endothelial cells. An ammonia plasma treatment was first used to graft amino groups on PTFE films. Subsequent reactions were performed to covalently bind human serum albumin (HSA) on the polymer surface and to load this protein with negative charges, which allows adsorbtion of VEGF onto HSA via strong electrostatic interactions. X-ray photoelectron spectroscopy (XPS) experiments along with surface derivatization strategies were performed between each synthesis step to ascertain the occurrence of the various molecules surface immobilization. Finally, the electrostatic binding of VEGF to the negatively charged HSA matrix was performed and validated by ELISA. Endothelial cell adhesion and migration experiments were carried out to validate the potential of this VEGF-containing biological construct to act as a proactive media toward the development of endothelial cells.

Improving biomaterial properties of collagen films by chemical modification
Tiller, J. C., G. Bonner, et al. (2001), Biotechnol Bioeng 73(3): 246-52.
Abstract: Films of bovine collagen were chemically modified with the goal of improving their biomaterial properties. The modified films were investigated with respect to their affinity to fibroblast and endothelial cells, as well as their antibacterial properties tested by adhesion of Staphylococcus aureus. Modifications that only change the net charge of collagen, such as acetylation, succinylation, and treatment with glutaraldehyde (all increase the negative charge), and amination with ethylenediamine (EDA), N,N-dimethyl-EDA (DMEDA), or butylamine (all increase the positive charge), did not dramatically alter the mammalian cell attachment to the film. In contrast, derivatization of collagen using methoxypoly(ethylene glycol) (PEG) diminished the attachment of fibroblasts by 98 +/- 1% and of endothelial cells by more than 99% compared to unmodified collagen. Moreover, the rate of growth of fibroblasts dropped by 97 +/- 1% and that of endothelial cells by 88 +/- 3% as a result of PEGylation of collagen. Adhesion of S. aureus cells also plummeted by 93 +/- 2% as a result of this PEGylation. With these antifouling properties, PEG-collagen may be a promising coating material for coronary stents. Subsequent derivatization of PEG-collagen with EDA or DMEDA abolished its mammalian cell-repelling ability, whereas bacterial cell repulsion was partially retained: for example, DMEDA-modified PEG-collagen exhibits up to a 5-fold lower bacterial adhesion than collagen. It is worth noting that a material that allows mammalian cell attachment but reduces bacterial adhesion could be useful as an implant or coating.

Improving the blood compatibility of polyurethane using carbon nanotubes as fillers and its implications to cardiovascular surgery
Meng, J., H. Kong, et al. (2005), J Biomed Mater Res A 74(2): 208-14.
Abstract: Blood compatibility has been an occlusion for biomaterials used in the cardiovascular system. In this work, a multiwalled carbon nanotubes-polyurethane composite (MWNT-PU) was prepared through a controlled co-precipitation. The surface chemical composition of treated carbon nanotubes was analyzed with XPS and the thermal behaviors of composite were characterized by DSC. The platelet adhesion and activation caused by the composite were evaluated by using SEM and flow cytometric analysis, respectively, and the disruption of red blood cells was analyzed through measuring the absorbance of free hemoglobin. The experimental results demonstrated that: (1) Multiwalled carbon nanotubes (MWNTs) with oxygen-containing functional groups could be well dispersed in polyurethane matrix through a controlled coprecipitation; (2) the composite surface displayed a significantly improved anticoagulant function, which can be indicative of the promising potentials of carbon nanotube-based materials in the implants and medical devices applied in blood-contacting environments.

Improving the interface between biomaterials and the blood. The gene therapy approach
Clowes, A. W. (1996), Circulation 93(7): 1319-20.

Impurity formation studies with peptide-loaded polymeric microspheres Part I. In vivo evaluation
Murty, S. B., B. C. Thanoo, et al. (2005), Int J Pharm 297(1-2): 50-61.
Abstract: The purpose of the present investigation was to assess the peptide related substances or impurities formed during incubation of drug loaded poly-(D,L-lactide-co-glycolide) (PLGA) and poly-(D,L-lactide) (PLA) microspheres under in vivo conditions. Sprague-Dawley rats were injected with separate batches of octreotide microspheres prepared by either an oil/water or oil/oil dispersion technique. At specified time points (days 14, 22, 30, and 41), animals were sacrificed and microsphere particles were recovered from the subcutaneous injection sites. The recovered particles were further extracted with 1:1 mixture of dimethylsulfoxide:dichloromethane for subsequent impurity analysis by HPLC and mass spectrometry. During incubation, the percentage purity of parent compound depended on the PLGA co-monomer ratio (e.g. 50:50, 85:15, and 100:0 glycolide:lactide ratios). After 41 days of incubation, for instance, octreotide area percentage by HPLC was determined to be approximately 47% for PLGA 50:50 microspheres, approximately 75% for PLGA 85:15 microspheres, and approximately 87% for PLA microspheres. Spectral analysis of particle extracts revealed the presence of peptide related substances with 58 m/z and 72 m/z units higher than the parent peptide m/z value. This indicated the presence of glycoyl and lactoyl covalent substitutions on the drug compound, resulting from chemical interaction between peptide amine groups and PLGA or PLA ester groups.

Impurity formation studies with peptide-loaded polymeric microspheres Part II. In vitro evaluation
Murty, S. B., D. H. Na, et al. (2005), Int J Pharm 297(1-2): 62-72.
Abstract: Since acylated peptide impurities were isolated from octreotide microspheres following incubation in an in vivo environment, the present investigation was undertaken to determine the dosage form dynamics responsible for facilitating acylation. In particular, microsphere batches made with poly(L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) 85:15 were studied for in vitro drug release, mass balance relationships, mass loss behavior, hydration uptake, and solid-state stability. Furthermore, native octreotide was incubated in a varying pH stability model (heat treated lactic acid solutions 42.5%, w/w) to determine the effects of acidity on impurity formation. From a review of the experimental results, the appearance of octreotide impurities or related substances occurred with the onset of polymeric mass loss. In fact, the significant formation of acylated peptide did not appear until >90% mass loss, which was observed at 14 days. It was surmised that because of water uptake, the hydrolytic cleavage of the polymeric backbone created an acidic microenvironment to facilitate the covalent coupling of peptide with polymer. The lactic acid solution stability model corroborated with greater evidence of acylation at pH 2.25 where the presence lactoyl (+72 m/z) derivatives of octreotide were confirmed by MALDI-TOF mass spectrometry.

In search of missing links in otology. I. Development of a collagen-based biomaterial
Goycoolea, M. V., D. C. Muchow, et al. (1991), Laryngoscope 101(7 Pt 1): 717-26.
Abstract: Experiments leading to the development and use of a biomaterial based on reconstituted collagen for use in tympanoplasty are presented. A stable, even membrane with optimal strength and an organized matrix of collagen protein strands has been obtained. Biocompatibility was documented by subcutaneous implantation, cytotoxicity with agar overlay, cell contact, and cell-growth inhibition studies. Experimental grafting in chinchillas with perforated tympanic membranes demonstrated that the collagen membrane performed well in all cases. Histopathological studies in chinchillas showed that the collagen membrane compared favorably with fascia grafts. Of significance is that: 1. The membrane has a matrix of microperforations that enhance tissue ingrowth, allow stable anchoring, and permit aeration of the middle ear cavity. 2. The membranes obtained are not exposed to aldehyde cross-linking; therefore, tissue reaction due to aldehydes is avoided.

In situ conformational analysis of fibrinogen adsorbed on Si surfaces
Tunc, S., M. F. Maitz, et al. (2005), Colloids Surf B Biointerfaces 42(3-4): 219-25.
Abstract: Fibrinogen is a major plasma protein. Previous investigations of structural changes of fibrinogen due to adsorption are mostly based on indirect evidence after its desorption, whereas our measurements were performed on fibrinogen in its adsorbed state. Specific enzyme-linked immunosorption experiments showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. Atomic force microscopy (AFM) investigations revealed the trinodular shape of fibrinogen molecules adsorbed on hydrophilic surfaces, whereas all of the molecules appeared globular on hydrophobic surfaces. The distribution of secondary structures in adsorbed fibrinogen was quantified by in situ Fourier-transform infrared (FTIR) analysis. Substrates of identical chemical bulk composition but different surface hydrophobicity permit direct comparison among them. Adsorption properties of fibrinogen are different for each degree of hydrophobicity. Although there is some increase of turn structure and decrease of beta-sheet structure, the secondary structure of adsorbed fibrinogen on hydrophilic surface turned out to be rather similar to that of the protein in solution phase with a major alpha-helix content. Hydrophilic surfaces exhibit superior blood compatibility as required for medical applications.

In situ formation of blends by photopolymerization of poly(ethylene glycol) dimethacrylate and polylactide
Zhang, K., C. G. Simon, Jr., et al. (2005), Biomacromolecules 6(3): 1615-22.
Abstract: Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.

In situ forming lactic acid based orthopaedic biomaterials: influence of oligomer chemistry on osteoblast attachment and function
Burdick, J. A., M. N. Mason, et al. (2001), J Biomater Sci Polym Ed 12(11): 1253-65.
Abstract: The ability of osteoblasts to attach and function normally on scaffolds fabricated from synthetic materials is essential for musculoskeletal tissue engineering applications. In this study, the osteoconductivity of polymer networks formed from multifunctional lactic acid oligomers was assessed. These oligomers form highly crosslinked networks via a photoinitiated polymerization, which provides potential advantages for many orthopaedic applications. Depending on the initial oligomer chemistry and the resultant polymer hydrophobicity, protein adsorption and osteoblast function varied significantly between the various lactic acid based polymer chemistries. Results were compared to control polymers of tissue culture polystyrene (TCPS) and 50:50 poly(lactic-co-glycolic acid) (PLGA). The viability of osteoblasts attached to poly(2EG10LA) and poly(2EG6LA) was close to the TCPS and PLGA after 7 and 14 days of culture, whereas cell viability was approximately 50% lower on poly(8EG6LA). Additionally, the alkaline phosphatase activity and mineralization of attached osteoblasts were similar on poly(2EG10LA) and PLGA, whereas these markers of bone formation were significantly lower for poly(2EG6LA) and poly(8EG6LA). For example, the alkaline phosphatase activity of rat calvarial osteoblasts attached to poly(2EG10LA) was 0.048 +/- 0.006 micromol mg(-1) protein-min, but only 0.030 +/- 0.003 micromol mg(-1) protein-min for osteoblasts attached to poly(8EG6LA) after 14 days of culture. Finally, osteoblasts were seeded onto three-dimensional scaffolds to demonstrate the applicability of the scaffolds for bone tissue engineering.

In situ polymerization of pyrrole in animal tissue in the formation of hybrid biomaterials
Khor, E., H. C. Li, et al. (1995), Biomaterials 16(8): 657-61.
Abstract: Porcine pericardium was impregnated with pyrrole or its derivative, sodium 4-(3-pyrrolyl)butanesulphonate (SPBS), by soaking the animal tissue in the monomer. Subsequent in situ chemical polymerization of the monomer-rich tissue using FeCl3 as initiator produced black polypyrrole-tissue hybrid biomaterials. The rate and extent of polymerization was found to be greater in 0.5 M acetic acid than in Hepes-buffered saline (HBS) and also greater for SPBS than for pyrrole. However, better tissue integrity was obtained for polymerization in HBS. Histological examination showed that the monomers do not permeate through the entire tissue, restricting polymerization to the surface layers of the tissue. The samples so formed do not exhibit detectable electrical conductivity.

In situ-gelling, erodible N-isopropylacrylamide copolymers
Lee, B. H. and B. Vernon (2005), Macromol Biosci 5(7): 629-35.
Abstract: Copolymers of N-isopropylacrylamide, 2-hydroxyethyl methacryl lactate [(HEMA)-lactate] and acrylic acid (AAc) were prepared with varying mole ratios of monomers to develop copolymers with gelation properties above a certain concentration for a bioerodible, in-situ gelling material. The copolymers formed gels in situ under physiological condition. The gelation temperature of the copolymers decreased as the HEMA-lactate content of the copolymers increased due to the hydrophobicity of HEMA-lactate, and increased as the AAc content increased due to the hydrophilicity of AAc. The gels redissolve at 37 degrees C as their LCSTs increase above 37 degrees C due to the hydrolysis of the HEMA-lactate pendant groups.

In utero repair of an experimental neural tube defect in a chronic sheep model using biomatrices
Eggink, A. J., L. A. Roelofs, et al. (2005), Fetal Diagn Ther 20(5): 335-40.
Abstract: OBJECTIVE: Persistent exposure of the unprotected spinal cord to amniotic fluid and the uterine wall can lead to progressive damage of neural tissue in case of a myelomeningocele (two-hit hypothesis). The aim of this study was to evaluate whether in utero repair of an experimental neural tube defect in a fetal lamb could protect neural tissue from secondary injury and save neurologic functions after birth. METHODS: In 19 fetal lambs, a neural tube defect was created at 79 days' gestation. In 12 lambs the defect was covered either with a novel, molecular defined collagen-based biocompatible and biodegradable matrix (UMC) or with a small intestinal submucosa (SIS) biomatrix (Cook) or by closing the skin over the defect. RESULTS: All lambs with the defect covered showed no or minor neurologic morbidity in contrast to the lambs with the defect uncovered in which major neurologic morbidity was seen. CONCLUSIONS: These results demonstrate that long-term exposure of the open spinal cord to the intrauterine environment can lead to damage of neural tissue and, consequently loss of neurologic functions and that coverage of the defect can lead to a better neurologic outcome. Furthermore, we could show that a UMC biomatrix and an SIS biomatrix are useful for in utero coverage of a surgically created neural tube defect in our model.

In vitro adherence of Enterococcus faecalis and Enterococcus faecium to plastic biomaterials
Joyanes, P., A. Pascual, et al. (1999), Clin Microbiol Infect 5(6): 382-386.

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Last Modified: 8 February 2006