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Methods: a comparative analysis of radiography, microcomputed tomography, and histology for bone tissue engineering
Hedberg, E. L., H. C. Kroese-Deutman, et al. (2005), Tissue Eng 11(9-10): 1356-67.
Abstract: This study focused on the assessment of radiography, microcomputed tomography, and histology for the evaluation of bone formation in a 15.0-mm defect in the rabbit radius after the implantation of a tissue-engineered construct. Radiography was found to be useful as a noninvasive method for obtaining images of calcified tissue throughout the time course of the experiment. With this method, however, image quality was low, making it difficult to obtain precise information about the location and quantity of the bone formed. Microcomputed tomography was used to create three-dimensional reconstructions of the bone (25-microm resolution). These reconstructions allowed for greater spatial resolution than the radiography, but did not allow for imaging of the implanted scaffold material or the surrounding, nonmineralized tissue. To visualize all materials within the defect area at the cellular level, histology was used. Histological analysis, however, is a destructive technique that did not allow for any further analysis of the samples. Each technique examined here has its own advantages and limitations, but each yields unique information regarding bone regeneration. It is only through the use of all three techniques that complete characterization of the bone growth and tissue/construct responses after implantation in vivo.

Methotrexate-loaded biodegradable polymeric micelles: preparation, physicochemical properties and in vitro drug release
Zhang, Y., T. Jin, et al. (2005), Colloids Surf B Biointerfaces 44(2-3): 104-9.
Abstract: Polymeric micelles based on amphiphilic diblock copolymers methoxy poly(ethylene glycol)-polylactide with various hydrophobic lengths were designed as carriers of poorly water-soluble anticancer drug methotrexate (MTX). Relationship between physicochemical characteristics of micelles and release behavior was explored. The critical micelle concentration was determined by fluorescence spectroscopy using 9-chloromethyl anthracene as fluorescence probe. Core-shell type polymeric micelles were prepared by free-surfactant dialysis technique. The mean size of micelles loaded with MTX was 50-200 nm with narrow polydispersity. Physicochemical properties of drug-loaded micelles were evaluated. In vitro release behavior of MTX was also investigated. MTX was continuously released from micelles and less than 50% MTX was released in 5 days. Release rate was dependent on chemical structures of micelles and enhanced by decreasing polylactide lengths.

Methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) copolymer micelles for formulation of hydrophobic drugs
Lee, H., F. Zeng, et al. (2005), Biomacromolecules 6(6): 3119-28.
Abstract: Six amphiphilic diblock copolymers based on methoxy poly(ethylene glycol) (MePEG) and poly(delta-valerolactone) (PVL) with varying hydrophilic and hydrophobic block lengths were synthesized via a metal-free cationic polymerization method. MePEG-b-PVL copolymers were synthesized using MePEG with Mn = 2000 or Mn = 5000 as the macroinitiator. 1H NMR and GPC analyses confirmed the synthesis of diblock copolymers with relatively narrow molecular weight distributions (Mn/Mw = 1.05-1.14). DSC analysis revealed that the melting temperatures (Tm) of the copolymers (47-58 degrees C) approach the Tm of MePEG as the PVL content is decreased. MePEG-b-PVL copolymer aggregates loaded with the hydrophobic anti-cancer drug paclitaxel were found to have effective mean diameters ranging from 31 to 970 nm depending on the composition of the copolymers. A MePEG-b-PVL copolymer of a specific composition was found to form drug-loaded micelles of 31 nm in diameter with a narrow size distribution and improve the apparent aqueous solubility of paclitaxel by more than 9000-fold. The biological activity of paclitaxel formulated in the MePEG-b-PVL micelles was confirmed in human MCF-7 breast and A2780 ovarian cancer cells. Furthermore, the biocompatibility of the copolymers was established in CHO-K1 fibroblast cells using a cell viability assay. The in vitro hydrolytic and enzymatic degradation of the micelles was also evaluated over a period of one month. The present study indicates that the MePEG-b-PVL copolymers are suitable biomaterials for hydrophobic drug formulation and delivery.

Methyl-DEAE-dextran: a candidate biomaterial
Zambito, Y., A. Baggiani, et al. (2004), Biomed Mater Eng 14(4): 411-7.
Abstract: The full quaternisation of DEAE-dextran was successfully attempted and an application of the quaternised product was suggested. Commercial DEAE-dextran was reacted with iodomethane at 60 degrees C in the presence of NaOH. The raw product was purified by dialysis, during which the iodide ion was replaced by chloride. N-methylation and O-methylation resulted from the reaction. A second methylation step produced no further changes in the molecule. Alkalimetry indicated the absence of amino groups in the methylated polymer molecule, thus testifying to a complete quaternisation. N-acetylcysteine (AcCy) was neutralised with the polymer in the hydroxide form, thus obtaining the methyl DEAE-dextran salt of AcCy (Me-DEAE-dextran/AcCy), whereby an ophthalmic formulation for the treatment of the dry eye syndrome was prepared. For comparison, the neutral AcCy salt of commercial DEAE-dextran (DEAE-dextran/AcCy) was prepared. The AcCy content in Me-DEAE-dextran/AcCy was higher than in DEAE-dextran/AcCy (23 vs 13%), while the viscosity of a solution containing the salt concentration corresponding to the therapeutic AcCy concentration (4%w/v) was lower with the former compared to the latter salt (20.5 vs 23.9 mPa s). Both solutions were ipotonic (245 mOsm/kg), whereas the commercial Tirocular is strongly hypertonic (900 mOsm/kg) and irritant.

Micelles from new biodegradable amphiphilic block copolymers containing PEG AND PCL
Signori, F., R. Solaro, et al. (2005), J Control Release 101(1-3): 379-81.

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation
Podesta, A., E. Ranucci, et al. (2005), J Nanosci Nanotechnol 5(3): 425-30.
Abstract: Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.

Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering
Le Nihouannen, D., L. L. Guehennec, et al. (2005), Biomaterials
Abstract: Calcium phosphate ceramics are currently used as bone graft substitutes in various types of clinical applications. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties. By combining these two biomaterials, new composite scaffolds were prepared. In this study, we attempt to analyse whether thrombin concentration in the fibrin glue could influence the properties of the composite. The association between fibrin glue and calcium phosphate ceramic granules was characterized at the ultra structural level. Micro and macroporous biphasic calcium phosphate ceramic granules with a diameter of 1-2mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) were associated to fibrin glue. The composites were observed by scanning and transmission electron microscopy and microcomputed tomography. Fibre thickness, porosity and homogeneity of the fibrin clot were modified by increased the thrombin concentration. Mixing fibrin glue with calcium phosphate granules (1:2) did not modify the microstructure of the fibrin clot in the composite. Nevertheless, thrombin interacted with the bioceramic by inducing the nucleation of crystalline precipitate at the ceramic/fibrin glue interface. Combining fibrin sealant and calcium phosphate ceramics could lead to new scaffolds for bone tissue engineering with the synergy of the properties of the two biomaterials.

Microbial cellulose--the natural power to heal wounds
Czaja, W., A. Krystynowicz, et al. (2006), Biomaterials 27(2): 145-51.
Abstract: Microbial cellulose (MC) synthesized in abundance by Acetobacter xylinum shows vast potential as a novel wound healing system. The high mechanical strength and remarkable physical properties result from the unique nanostructure of the never-dried membrane. This article attempts to briefly summarize the recent developments and applications of MC in the emerging field of novel wound dressings and skin substitutes. It considers the properties of the synthesized material, its clinical performance, as well as progress in the commercialization of MC for wound care products. Efficient and inexpensive fermentation techniques, not presently available, will be necessary to produce large quantities of the polymer.

Microbial polyhydroxyalkanoates (PHAs): an emerging biomaterial for tissue engineering and therapeutic applications
Sudesh, K. (2004), Med J Malaysia 59 Suppl B: 55-6.
Abstract: Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates (PHAs) offer the most diverse range of thermal and mechanical properties. Of particular interest are the PHAs that contain 4-hydroxybutyrate such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB) and poly(4-hydroxybutyrate) [P(4HB)]. These polyesters can only be synthesized by a few types of bacteria, among which Comamonas acidovorans has the most efficient metabolic pathways to channel 4HB monomers. The resulting polyesters are bioabsorbable and are being developed as a new biomaterial for medical applications. By controlling the molar ratio of the monomers, it is possible to produce materials that are as tough and elastic as rubber.

Microcalcifications associated with breast cancer: an epiphenomenon or biologically significant feature of selected tumors?
Morgan, M. P., M. M. Cooke, et al. (2005), J Mammary Gland Biol Neoplasia 10(2): 181-7.
Abstract: Radiographic mammary calcifications occur in 30-50% of breast cancers and constitute one of the most important diagnostic markers of both benign and malignant lesions of the breast. The presence of oxalate-type microcalcification appears to be a reliable criterion in favor of the benign nature of the lesion or, at most, of a lobular carcinoma in situ. In contrast, calcium hydroxyapatite (HA) crystals are associated with both benign and malignant breast tumors. Although the diagnostic value of microcalcifications in breast cancer is of great importance, the genesis of these calcifications is unclear. Despite numerous histological ultrastructure studies of HA deposits in breast carcinomas, to date there have been limited investigations of the potential role of these crystals in breast cancer. We review the literature examining the biological effects of HA crystals in breast cancer cell lines, specifically the mechanism of HA-induced mitogenesis and upregulation of gene expression.

Microcalorimetry of the adsorption of lysozyme onto polymeric substrates
Lee, V. A., R. G. Craig, et al. (2005), J Colloid Interface Sci 288(1): 6-13.
Abstract: The conformation of blood proteins adsorbed on biomaterial surfaces probably plays a significant role in the biocompatibility of blood-contacting implants. This paper reports heats of adsorption of a globular protein, lysozyme, onto three uncharged polymeric substrates. Variations in heats among substrates reflect differences in the lysozyme/substrate interaction as well as the possibility of substrate-dependent conformations. In each case, a series of plateaus appeared in the adsorption isotherm with increasing concentration. In the cases of two substrates, polystyrene and poly(styrene-co-butyl methacrylate), endothermic discontinuities appeared at rises between plateaus. It is proposed that the step pattern in each isotherm reflects distinct conformational states of lysozyme on the substrate surfaces. Endothermic discontinuities may be latent heats associated with change to a more stable conformation after initial adsorption. The absence of discontinuities in the case of poly(styrene-co-allyl alcohol) suggests structural transitions occur to a smaller extent on this material and results in conformations different from those which occur when lysozyme is adsorbed by polystyrene or by poly(styrene-co-butyl methacrylate).

Microcapsules prepared with different biomaterials to immobilize GDNF secreting 3T3 fibroblasts
Ponce, S., G. Orive, et al. (2005), Int J Pharm 293(1-2): 1-10.
Abstract: Cell microencapsulation represents a promising tool for the treatment of many central nervous system (CNS) diseases such as Parkinson's disease. In this technology, cells are surrounded by a semipermeable membrane which protects them from mechanical stress and isolates them from host's immune response. However, if the future clinical application of this strategy is wanted, many challenges remain including the improvement of the mechanical resistance of the microcapsules and the optimization of the intracapsular microenvironment conditions. In this way, the selection of the matrix is essential because the morphological and the physiological behavior of the cells depend on the interactions between the matrix and the enclosed cells. Assuming these considerations, three types of microcapsules elaborated with four different polymers: alginate, cellulose sulfate, agarose and pectin have been fabricated and compared in order to evaluate some key properties such as morphology, size and mechanical stability. Furthermore, GDNF secreting Fischer rat 3T3 fibroblasts were immobilized in each type of capsule and the viability and neurotrophic factor release was determinated. Results showed that the alginate and pectin microcapsules were the most resistant devices, maintaining an adequate microenvironment for the enclosed cells. In contrast, cells entrapped in alginate-cellulose sulfate matrices presented the lowest mechanical resistance, cell viability and GDNF production.

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips
Nagata, H., M. Tabuchi, et al. (2005), Electrophoresis 26(11): 2247-53.
Abstract: Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.

Microcomputer-based interactive tracking of blood cells at biomaterial surfaces
Capson, D. W., R. A. Maludzinski, et al. (1989), IEEE Trans Biomed Eng 36(8): 860-4.
Abstract: A microcomputer-based system for analyzing the motion of human platelets and leukocytes at synthetic surfaces from a sequence of video frames on tape is described. The software is designed to provide convenient interaction with an operator to reduce the burden of manual analysis. In addition, the system computes and stores the cell movement data on disk for subsequent statistical analysis. Measurement include the number and nature of cell-to-surface collisions, residence times, and distances traveled.

Microcrystalline chitosan is ineffective to decrease plasma lipids in both apolipoprotein E epsilon 4 carriers and non-carriers: a long-term placebo-controlled trial in hypercholesterolaemic volunteers
Lehtimaki, T., S. Metso, et al. (2005), Basic Clin Pharmacol Toxicol 97(2): 98-103.
Abstract: Chitosan is a deacetylated product of chitin. Microcrystalline form of chitosan has a large adsorption area claimed to decrease gastrointestinal absorption of cholesterol. However, the long-term effect of chitosan on plasma lipids is variable, the averaged influence being negligible or lacking in mildly-to-moderately hypercholesterolaemic (4.8-6.8 mmol/l) subjects. We evaluated whether this variation and inefficacy depend on apolipoprotein E genotype. 130 middle-aged, otherwise healthy men (n=55) and women (n=75) were randomized into two treatment groups for a 7 month trial. During a 1 month run-in period all participants received placebo. Subsequently, one half first took placebo twice daily for 3 months and then 1.2 g chitosan twice daily for 3 months, and the other half vice versa in a cross-over way. Altogether 84 participants completed the study. Plasma lipids and glucose were determined at the end of each phase of the study, and all subjects undergone to the cross-over phases were apolipoprotein E genotyped. Chitosan altered plasma total, low- and high density cholesterol, triglycerides, and blood glucose in neither apolipoprotein E epsilon 4 allele carriers (n=29) nor non-carriers (n=55), compared to placebo. In conclusions, chitosan is ineffective to decrease plasma lipids in apolipoprotein E epsilon 4 carrier and non-carrier phenotypes with mildly-to-moderately increased plasma cholesterol.

Microencapsulated chitosan nanoparticles for lung protein delivery
Grenha, A., B. Seijo, et al. (2005), Eur J Pharm Sci 25(4-5): 427-37.
Abstract: It has already been demonstrated that spray drying is a very valuable technique for producing dry powders adequate for pulmonary delivery of drugs. We have developed chitosan/tripolyphosphate nanoparticles that promote peptide absorption across mucosal surfaces. The aim of this work was to microencapsulate protein-loaded chitosan nanoparticles using typical aerosol excipients, such as mannitol and lactose, producing microspheres as carriers of protein-loaded nanoparticles to the lung. The results showed that the obtained microspheres are mostly spherical and possess appropriate aerodynamic properties for pulmonary delivery (aerodynamic diameters between 2 and 3 microm, apparent density lower than 0.45 g/cm3). Moreover, microspheres morphology was strongly affected by the content of chitosan nanoparticles. These nanoparticles show a good protein loading capacity (65-80%), providing the release of 75-80% insulin within 15 min, and can be easily recovered from microspheres after contact with an aqueous medium with no significant changes in their size and zeta potential values. Therefore, this work demonstrated that protein-loaded nanoparticles could be successfully incorporated into microspheres with adequate characteristics to reach the deep lung, which after contact with its aqueous environment are expected to be able to release the nanoparticles, and thus, the therapeutic macromolecule.

Microgel-based engineered nanostructures and their applicability with template-directed layer-by-layer polyelectrolyte assembly in protein encapsulation
Shenoy, D. B. and G. B. Sukhorukov (2005), Macromol Biosci 5(5): 451-8.
Abstract: A novel strategy for the fabrication of microcapsules is elaborated by employing biomacromolecules and a dissolvable template. Calcium carbonate (CaCO(3)) microparticles were used as sacrificial templates for the two-step deposition of polyelectrolyte coatings by surface controlled precipitation (SCP) followed by the layer-by-layer (LbL) adsorption technique to form capsule shells. When sodium alginate was used for inner shell assembly, template decomposition with an acid resulted in simultaneous formation of microgel-like structures due to calcium ion-induced gelation. An extraction of the calcium after further LbL treatment resulted in microcapsules filled with the biopolymer. The hollow as well as the polymer-filled polyelectrolyte capsules were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and scanning force microscopy (SFM). The results demonstrated multiple functionalities of the CaCO(3) core - as supporting template, porous core for increased polymer accommodation/immobilization, and as a source of shell-hardening material. The LbL treatment of the core-inner shell assembly resulted in further surface stabilization of the capsule wall and supplementation of a nanostructured diffusion barrier for encapsulated material. The polymer forming the inner shell governs the chemistry of the capsule interior and could be engineered to obtain a matrix for protein/drug encapsulation or immobilization. The outer shell could be used to precisely tune the properties of the capsule wall and exterior. [Diagram: see text] Confocal laser scanning microscopy (CLSM) image of microcapsules (insert is after treating with rhodamine 6G to stain the capsule wall).

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials
Richards, R. G., I. ap Gwynn, et al. (1995), Cell Biol Int 19(12): 1015-24.
Abstract: Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.

Micropatterned structures for studying the mechanics of biological polymers
Schek, H. T., 3rd and A. J. Hunt (2005), Biomed Microdevices 7(1): 41-6.
Abstract: Studying the mechanics of nanometer-scale biomolecules presents many challenges; these include maintaining light microscopy image quality and avoiding interference with the laser used for mechanical manipulation, that is, optical tweezers. Studying the pushing forces of a polymerizing filament requires barriers that meet these requirements and that can impede and restrain nanoscale structures subject to rapid thermal movements. We present a flexible technique that meets these criteria, allowing complex barrier geometries with undercut sidewall profiles to be produced on #1 cover glass for the purpose of obstructing and constraining polymerizing filaments, particularly microtubules. Using a two-layer lithographic process we are able to separate the construction of the primary features from the construction of a depth and shape-controlled undercut. The process can also be extended to create a large uniform gap between an SU-8 photoresist layer and the glass substrate. This technique can be easily scaled to produce large quantities of shelf-stable, reusable microstructures that are generally applicable to microscale studies of the interaction of cellular structures with defined microscale features.

Micropatterning of proteins and mammalian cells on biomaterials
Wang, Y. C. and C. C. Ho (2004), Faseb J 18(3): 525-7.
Abstract: Controlling the spatial organization of cells is vital in engineering tissues that require precisely defined cellular architectures. For example, functional nerves or blood vessels form only when groups of cells are organized and aligned in very specific geometries. Yet, scaffold designs incorporating spatially defined physical cues such as microscale surface topographies or spatial patterns of extracellular matrix to guide the spatial organization and behavior of cells cultured in vitro remain largely unexplored. Here we demonstrate a new approach for controlling the spatial organization, spreading, and orientation of cells on two micropatterned biomaterials: chitosan and gelatin. Biomaterials with grooves of defined width and depth were fabricated using a two-step soft lithography process. Selective attachment and spreading of cells within the grooves was ensured by covalently modifying the plateau regions with commercially available protein resistant triblock copolymers. Precise spatial control over cell spreading and orientation has been observed when human microvascular endothelial cells are cultured on these patterned biomaterials, suggesting the potential of this technique in creating tissue culture scaffolds with defined chemical and topographical features.

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Last Modified: 8 February 2006