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Changes in mechanical properties and cellularity during long-term culture of collagen fiber ACL reconstruction scaffolds
Caruso, A. B. and M. G. Dunn (2005), J Biomed Mater Res A 73(4): 388-97.
Abstract: Resorbable scaffolds for anterior cruciate ligament (ACL) reconstruction should provide temporary mechanical function then gradually breakdown while promoting matrix synthesis by local cells. Crosslinking influences collagen's mechanical properties, degradation rate, and interactions with cells. Our objective was to compare the effects of different crosslinkers on cellularity and mechanical properties during long-term (8 week) culture of collagen fiber scaffolds. Fibers were fabricated from an acid-insoluble dispersion of bovine dermal collagen and crosslinked with either ultraviolet irradiation (UV; a physical crosslinker) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC; a chemical crosslinker). Scaffolds consisted of 50 fibers bundled in parallel. Initial attachment of fibroblasts was similar on both scaffolds; however, from 1 to 8 weeks in culture, UV-crosslinked scaffolds had significantly more cells attached than EDC-crosslinked scaffolds. The initial breaking load (3.50 N) and stiffness (2.23 N/mm) of EDC-crosslinked scaffolds were significantly greater than those of UV-crosslinked scaffolds (2.32 N; 1.21 N/mm) and were unaffected by long-term fibroblast culture. In contrast, the load-bearing capacity of fibroblast-seeded UV-crosslinked scaffolds decreased 60% to 0.91 N after 8 weeks in culture. EDC-crosslinked scaffolds maintained strength and moderate cellularity; UV-crosslinked scaffolds, in contrast, were highly cellular, but had poor mechanical properties that decreased during culture. These in vitro results suggest that collagen fiber scaffolds crosslinked with EDC may be more suitable for ACL reconstruction than those crosslinked with UV.

Changes in platelet aggregation during cardiopulmonary bypass: comparison of poly-2-methoxyethylacrylate and heparin as a circuit coating material
Izuha, H., M. Hattori, et al. (2005), J Artif Organs 8(1): 41-6.
Abstract: At present, there are various biomaterials that have high biocompatibility. In particular, there are many types of coated circuits in cardiopulmonary bypass (CPB) systems. However, only a few clinical studies have investigated platelet aggregation caused by these coated circuits. In this study, a CPB system coated with poly-2-methoxyethylacrylate (X coating) was used to ascertain whether platelet aggregation could be suppressed during CPB, and a comparison was made between X coating and ordinary (covalently bonded) heparin coating. The subjects were 19 adult patients who were scheduled to undergo valve replacement or valvuloplasty. They were divided into two groups: group X (X coating) and group H (heparin coating). The platelet aggregation threshold index (PATI, grading curve) and beta-thromboglobulin and plalelet factor IV levels were assessed preoperatively (control), 5 min after heparin administration, 10 and 60 min after the start of CPB, and 0 and 2 h after the end of CPB. The results indicated that platelet aggregation was reduced during CPB and that platelets were activated. The changes in platelet aggregation associated with the X coating were shown to be similar to those associated with heparin coating.

Characterisation of human knee meniscus cell phenotype
Verdonk, P. C., R. G. Forsyth, et al. (2005), Osteoarthritis Cartilage 13(7): 548-60.
Abstract: OBJECTIVE: Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype. MATERIALS AND METHODS: Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated. RESULTS: The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1. CONCLUSION: These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.

Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial
Bobyn, J. D., G. J. Stackpool, et al. (1999), J Bone Joint Surg Br 81(5): 907-14.
Abstract: We have studied the characteristics of bone ingrowth of a new porous tantalum biomaterial in a simple transcortical canine model using cylindrical implants 5 x 10 mm in size. The material was 75% to 80% porous by volume and had a repeating arrangement of slender interconnecting struts which formed a regular array of dodecahedron-shaped pores. We performed histological studies on two types of material, one with a smaller pore size averaging 430 microm at 4, 16 and 52 weeks and the other with a larger pore size averaging 650 microm at 2, 3, 4, 16 and 52 weeks. Mechanical push-out tests at 4 and 16 weeks were used to assess the shear strength of the bone-implant interface on implants of the smaller pore size. The extent of filling of the pores of the tantalum material with new bone increased from 13% at two weeks to between 42% and 53% at four weeks. By 16 and 52 weeks the average extent of bone ingrowth ranged from 63% to 80%. The tissue response to the small and large pore sizes was similar, with regions of contact between bone and implant increasing with time and with evidence of Haversian remodelling within the pores at later periods. Mechanical tests at four weeks indicated a minimum shear fixation strength of 18.5 MPa, substantially higher than has been obtained with other porous materials with less volumetric porosity. This porous tantalum biomaterial has desirable characteristics for bone ingrowth; further studies are warranted to ascertain its potential for clinical reconstructive orthopaedics.

Characteristics of felodipine-located poly(epsilon-caprolactone) microspheres
Kim, B. K., S. J. Hwang, et al. (2005), J Microencapsul 22(2): 193-203.
Abstract: Felodipine-loaded poly (epsilon-caprolactone) (PCL) microspheres were prepared by two methods, the conventional emulsion solvent evapouration method and the quenching method. The aim of this work was to investigate the effects of process parameters such as emulsion type, drug loading, molecular-weight of the polymer, types of emulsion stabilizer and dispersed phase solvents, as well as preparation methods. The results show that, when conventional emulsion solvent evapouration method was used, the o/w-method produced smaller mean size and higher encapsulation efficiency compared with the o/o-method. The encapsulation efficiencies increased with an increase in the molecular weight and a decrease in crystallinity of PCL. The size of microspheres varied with the type of emulsion stabilizer used, smaller microspheres with PVA and narrow size distribution with Pol 237. The water solubility of the dispersed phase solvent was one of the critical factors in controlling the encapsulation efficiency and microsphere mean size. When water-soluble solvents such as acetonitrile and ethyl formate were used, the encapsulation efficiencies decreased due to higher evapouration rate. When quenching methods were used, in contrast to the conventional emulsion solvent evapouration method, very narrowly size-distributed but bigger microspheres were obtained.

Characteristics of interpolyelectrolyte complexes of Eudragit E 100 with sodium alginate
Moustafine, R. I., V. A. Kemenova, et al. (2005), Int J Pharm 294(1-2): 113-20.
Abstract: With a view to the application in oral drug delivery formulations, the possibility to form interpolyelectrolyte complexes (IPEC) of Eudragit E 100 (EE) with sodium alginate (AL) was investigated, employing turbidimetry, apparent viscosity measurements, FT-IR and elementary analysis. The interaction or binding ratio of a unit molecule of AL with EE was largely affected by the pH value of the media, showing a change from 1.5:1 to 1:1.25 (0.66

Characteristics of Poly-L-Ornithine-coated alginate microcapsules
Darrabie, M. D., W. F. Kendall, Jr., et al. (2005), Biomaterials 26(34): 6846-52.
Abstract: Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean +/- SEM diameter of PLL-coated microcapsules increased from 718+/-17 to 821 +/- 17 microm (p < 0.05) during 14 days incubation at 37 degrees C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7 +/- 4.9%) compared to 3.1 +/- 2.05% for PLO capsules (p < 0.0001,n = 6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.

Characterization and adsorption properties of eggshells and eggshell membrane
Tsai, W. T., J. M. Yang, et al. (2006), Bioresour Technol 97(3): 488-93.
Abstract: The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of fibrous proteins. From the isotherm data of methylene blue at 25 degrees C, the Freundlich model yielded a somewhat better fit than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0mg/g in aqueous medium, which was attributed to their poor pore properties.

Characterization and assessment of a novel poly(ethylene oxide)/polyurethane composite hydrogel (Aquavene) as a ureteral stent biomaterial
Gorman, S. P., M. M. Tunney, et al. (1998), J Biomed Mater Res 39(4): 642-9.
Abstract: The effective long-term use of indwelling ureteral stents is often hindered by the formation of encrusting deposits which may cause obstruction and blockage of the stent. Development of improved ureteral stent biomaterials capable of preventing or reducing encrustation is therefore particularly desirable. In this study, the suitability as a ureteral stent biomaterial of Aquavene, a novel poly(ethylene oxide)/polyurethane composite hydrogel was compared with that of silicone and polyurethane, two materials widely employed in ureteral stent manufacture. Examination of Aquavene in dry and hydrated states by confocal laser scanning microscopy, scanning electron microscopy, and atomic force microscopy showed the presence of numerous channels within a cellular matrix structure. The channel size increased considerably to as much as 10 microm in diameter in the hydrated state. Aquavene provided superior resistance to encrustation and intraluminal blockage over a 24-week period in a simulated urine flow model. Unobstructed urine flow continued with Aquavene at 24 weeks, whereas silicone and polyurethane stents became blocked with encrustation at 8 and 10 weeks, respectively. Weight loss within Aquavene on the order of 9% (w/w) over the 24-week flow period indicates that extraction of the noncrosslinked poly(ethylene oxide) hydrogel may be responsible for the prevention of encrustation blockage of this biomaterial. In the dry state, Aquavene was significantly harder than either silicone or polyurethane, as shown by Young's modulus, and rapidly became soft on hydration. These additional properties of Aquavene would facilitate ease of stent insertion in the dry state past obstructions in the ureter and provide improved patient comfort on subsequent biomaterial hydration in situ. Aquavene is a promising candidate for use in the urinary tract, as it is probable that effective long-term urine drainage would be maintained in vivo. Further evaluation of this novel biomaterial is therefore warranted.

Characterization and biocompatibility of organogels based on L-alanine for parenteral drug delivery implants
Motulsky, A., M. Lafleur, et al. (2005), Biomaterials 26(31): 6242-53.
Abstract: The development of simple and efficient drug delivery systems for the sustained release of peptides/proteins and low molecular weight hydrophilic molecules is an ongoing challenge. The purpose of this work was to prepare and characterize novel biodegradable in situ-forming implants obtained via the self-assembly of L-alanine derivatives in pharmaceutical oils. Six different amphiphilic organogelators based on L-alanine were synthesized. These derivatives could successfully gel various vegetable and synthetic oils approved for parenteral administration. Gelation was thermoreversible, and phase transition temperatures depended on gelator structure, concentration and solvent. Hydrogen bonds and van der Waals interactions were shown to be the main forces implicated in network formation. Selected formulations were then injected subcutaneously in rats for preliminary assessment of biocompatibility. Histopathological analysis of the surrounding tissues revealed mild, chronic inflammation and an overall good biocompatibility profile of the implants over the 8 wk evaluation period. This study demonstrates that in situ-forming organogels represent a potentially promising platform for sustained drug delivery.

Characterization and bond strength of electrolytic HA/TiO2 double layers for orthopaedic applications
Lin, C. M. and S. K. Yen (2005), J Mater Sci Mater Med 16(10): 889-97.
Abstract: Insufficient bonding of juxtaposed bone to an orthopaedic/dental implant could be caused by material surface properties that do not support new bone growth. For this reason, fabrication of biomaterials surface properties, which support osteointegration, should be one of the key objectives in the design of the next generation of orthopaedic/dental implants. Titanium and titanium alloy have been widely used in several bioimplant applications, but when implanted into the human body, these still contain some disadvantages, such as poor osteointegration (forming a fibrous capsule), wear debris and metal ion release, which often lead to clinical failure. Electrolytic hydroxyapatite/titanium dioxide (HA/TiO2) double layers were successfully deposited on titanium substrates in TiCl4 solution and subsequently in the mixed solution of Ca(NO3)2 and NH4H2PO4, respectively. After annealing at 300 degrees C for 1 h in the air, the coated specimens were evaluated by dynamic cyclic polarization tests, immersion tests, tensile tests, surface morphology observations, XRD analyses and cells culture. The adhesion strength of the HA coating were improved by the intermediate coating of TiO2 from 11.3 to 46.7 MPa. From cell culture and immersion test results, the HA/TiO2 coated specimens promoted not only cells differentiation, but also appeared more bioactive while maintaining non-toxicity.

Characterization and bond strength of electrolytic HA/TiO2 double layers for orthopedic applications
Lin, C. M. and S. K. Yen (2004), J Mater Sci Mater Med 15(11): 1237-46.
Abstract: Insufficient bonding of juxtaposed bone to an orthopedic/dental implant could be caused by material surface properties that do not support new bone growth. For this reason, fabrication of biomaterials surface properties, which support osteointegration, should be one of the key objectives in the design of the next generation of orthopedic/dental implants. Titanium and titanium alloy have been widely used in several bioimplant applications, but when implanted into the human body, these still contain some disadvantages, such as poor osteointegration (forming a fibrous capsule), wear debris and metal ion release, which often lead to clinical failure. Electrolytic hydroxyapatite/titanium dioxide (HA/TiO2) double layers were successfully deposited on titanium substrates in TiCl4 solution and subsequently in the mixed solution of Ca(NO3)2 and NH4H2PO4, respectively. After annealing at 300 degrees C for 1 h in the air, the coated specimens were evaluated by dynamic cyclic polarization tests, immersion tests, tensile tests, surface morphology observations, XRD analyses and cells culture. The adhesion strength of the HA coating were improved by the intermediate coating of TiO2 from 11.3 to 46.7 MPa. From cell culture and immersion test results, the HA/TiO2 coated specimens promoted not only cells differentiation, but also appeared more bioactive while maintaining non-toxicity.

Characterization and degradation of functionalized chitosan with glycidyl methacrylate
Flores-Ramirez, N., E. A. Elizalde-Pena, et al. (2005), J Biomater Sci Polym Ed 16(4): 473-88.
Abstract: The synthesis, characterization and degradation of a hybrid chitosan (CTS)/glycidyl methacrylate (GMA) material are reported. These versatile materials (natural-synthetic materials) are potential candidates for dental restoratives. All materials were characterized by infrared spectroscopy (FT-IR), X-ray diffraction and thermal (DSC) analysis. Particular attention was paid to the thermal stability and chemical resistance of the hybrid CTS materials. From dynamical rheological tests, it was concluded that CTS-GMA solutions behave as physical hydrogels. These pH-sensitive gels are an example of stimuli-responsive polymers, also known as 'smart polymers'.

Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial
Cook, A. D., J. S. Hrkach, et al. (1997), J Biomed Mater Res 35(4): 513-23.
Abstract: The design of biomaterials containing specific ligands on the surface offers the possibility of creating materials that can interact with and potentially control mammalian cell behavior. Biodegradable materials further provide the significant advantage that the polymer will disappear in vivo, obviating long-term negative tissue responses as well as the need for retrieval. In earlier studies we synthesized and characterized arginine-glycine-aspartic acid (RGD) peptide-modified poly(lactic acid-co-lysine) (PLAL). In this study, both bulk properties and surface features have been characterized, with a focus on surface analysis as a means of interpreting observed changes in cell behavior. Bulk peptide attachments were performed using 1,1'-carbonyldiimidazole (CDI). Amino groups were measured using colorimetric assays and X-ray photoelectron spectroscopy (XPS). Peptides were measured by incorporating iodine into the peptide as a distinct elemental marker for use with XPS. Typical samples contained 13 +/- 4 pmol/cm2 of amino groups and 4 +/- 0.2 pmol/ cm2 of peptides, as calculated from XPS measurements of nitrogen and iodine. The wettability and crystallinity of the samples were determined by contact angles and differential scanning calorimetry, respectively. Wettability and crystallinity were not altered by the incorporation of lysine or peptides. After incubating bovine aortic endothelial (BAE) cells for 4 h on surfaces with RGD-containing peptides, the mean spread cell area increased from 77 +/- 2 microns2 to 405 +/- 29 microns2 compared to 116 +/- 11 microns2 on poly(lactic acid), 87 +/- 4 microns2 on PLAL, and 105 +/- 4 microns2 on surfaces with RDG-containing (control) peptides. The significance of this work is that the first synthetic interactive, resorbable biomaterial has been developed, and use of this material to control cell behavior has been demonstrated.

Characterization and evaluation of whey protein-based biofilms as substrates for in vitro cell cultures
Gilbert, V., M. Rouabhia, et al. (2005), Biomaterials 26(35): 7471-80.
Abstract: Whey proteins-based biofilms were prepared using different plasticizers in order to obtain a biomaterial for the human keratinocytes and fibroblasts in vitro culture. The film properties were evaluated by Fourier Transform Infrared Spectroscopy (FTIR) technique and mechanical tests. A relationship was found between the decrease of intermolecular hydrogen bond strength and film mechanical behavior changes, expressed by a breaking stress and Young modulus values diminishing. These results allow stating that the film molecular configuration could induce dissimilarities in its mechanical properties. The films toxicity was assessed by evaluating the cutaneous cells adherence, growth, proliferation and structural stratification. Microscopic observation demonstrated that both keratinocytes and fibroblasts adhered to the biofilms. The trypan blue exclusion test showed that keratinocytes grew at a significantly high rate on all the biofilms. Structural analysis demonstrated that keratinocytes stratified when cultured on the whey protein-based biofilms and gave rise to multi-layered epidermal structures. The most organized epidermis was obtained with whey protein isolate/DEG biofilm. This structure had a well-organized basal layer under supra-basal and corneous layers. This study demonstrated that whey proteins, an inexpensive renewable resource which can be obtained readily, were non-toxic to cutaneous cells and thus they could be useful substrates for a variety of biomedical applications, including tissue engineering.

Characterization and optimization of experimental variables within a reproducible bladder encrustation model and in vitro evaluation of the efficacy of urease inhibitors for the prevention of medical device-related encrustation
Jones, D. S., J. Djokic, et al. (2006), J Biomed Mater Res B Appl Biomater 76(1): 1-7.
Abstract: This study presents a reproducible, cost-effective in vitro encrustation model and, furthermore, describes the effects of components of the artificial urine and the presence of agents that modify the action of urease on encrustation on commercially available ureteral stents. The encrustation model involved the use of small-volume reactors (700 mL) containing artificial urine and employing an orbital incubator (at 37 degrees C) to ensure controlled stirring. The artificial urine contained sources of calcium and magnesium (both as chlorides), albumin and urease. Alteration of the ratio (% w/w) of calcium salt to magnesium salt affected the mass of encrustation, with the greatest encrustation noted whenever magnesium was excluded from the artificial urine. Increasing the concentration of albumin, designed to mimic the presence of protein in urine, significantly decreased the mass of both calcium and magnesium encrustation until a plateau was observed. Finally, exclusion of urease from the artificial urine significantly reduced encrustation due to the indirect effects of this enzyme on pH. Inclusion of the urease inhibitor, acetohydroxamic acid, or urease substrates (methylurea or ethylurea) into the artificial medium markedly reduced encrustation on ureteral stents. In conclusion, this study has described the design of a reproducible, cost-effective in vitro encrustation model. Encrustation was markedly reduced on biomaterials by the inclusion of agents that modify the action of urease. These agents may, therefore, offer a novel clinical approach to the control of encrustation on urological medical devices. (c) 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006.

Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes
Estrada, L. E., G. R. Dodge, et al. (2001), Osteoarthritis Cartilage 9(2): 169-77.
Abstract: OBJECTIVE: The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the biochemical and histological characteristics of the resulting tissue. DESIGN: Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for > or = 8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0 x 10(7) cells/ml) in DMEM with 10% FBS, 50 microg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60 mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing(3)H-thymidine incorporation and for their collagen production employing biosynthetic labeling with(14)C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR. RESULTS: Within 24 h of culture, isolated chondrocytes organized into well-formed clusters and in 2 weeks formed structures with gross appearance and consistency similar to those of natural cartilage. The wet weight of the tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of(3)H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was not detected by Western blot over the 10-week period. High levels of type II and type X collagen gene expression were demonstrated by RT-PCR. CONCLUSION: These studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.

Characterization of a chitosanase isolated from a commercial ficin preparation
Chiang, C. L., Y. M. Chang, et al. (2005), J Agric Food Chem 53(19): 7579-85.
Abstract: A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).

Characterization of a new stationary phase based on microwave immobilized polybutadiene on titanium oxide-modified silica
Morais, L. S. and I. C. Jardim (2005), J Chromatogr A 1073(1-2): 127-35.
Abstract: Titanium oxide-modified silica was prepared by reaction of silica with titanium tetrabutoxide and then was used as support in the preparation of stationary phases with self-immobilized polybutadiene (PBD) and PBD immobilized through microwave radiation. Chromatographic performance of the stationary phases was evaluated in terms of the efficiency (plates/m), asymmetry (A(s)), retention factor (k) and resolution (R(s)) of two standard sample mixtures, one of then containing the basic compound N,N-dimethylaniline. A microwave irradiation of 30 min at 520 W gave the best efficiency (86,500 N m(-1)), greater than that of a 6-day self immobilized phase (69,500 N m(-1)). Self-immobilized stationary phases prepared with bare silica were also studied for comparison. These resulted in lower chromatographic performance, 43,800 N m(-1), when compared to the self-immobilized phase prepared with titanized silica.

Characterization of biomaterial surfaces: ATR-FTIR, potentiometric and calorimetric analysis
Barbucci, R., M. Casolaro, et al. (1992), Clin Mater 11(1-4): 37-51.
Abstract: The usefulness of attenuated total reflection-Fourier transform infrared spectroscopy, potentiometry and calorimetry in surface characterization of biomaterials is discussed in this paper. Some examples of biomedical materials characterized by using these different techniques are also reported.

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Last Modified: 8 February 2006