bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of macroporous biodegradable poly(L‐lactide‐co‐ϵ‐caprolactone) foams and characterization by mercury intrusion porosimetry, image analysis, and impedance spectroscopy
Authors: Maquet V. , Blacher S. , Pirard R. , Pirard J.‐P. , Vyakarnam M. N. , Jérôme R.
Published: July 2003

Two poly(L‐lactide‐co‐ϵ‐caprolactone) random copolymers containing 5 and 40 mol % of ϵ‐CL, namely P(LA‐co‐CL5) and P(LA‐co‐CL40), respectively, have been made macroporous by freeze‐drying solutions in dimethylcarbonate. Most of the freeze‐dried foams, prepared by varying polymer concentration and cooling rate, exhibited two main pore populations: (1) longitudinally oriented tube‐like macropores with diameters ≥100 μm, and (2) interconnected micropores (10–100 μm). Pore characteristics, including macropore density, mean diameter, and interdistance, as well as micropore density, area, and shape, were determined by image analysis of scanning electron micrographs in order to study the influence of processing and formulation parameters on foam structure and properties. The pore orientation and the 3‐D texture also were studied by image analysis and impedance spectroscopy. In the case of the P(LA‐co‐CL5), the macropore diameter increased with the cooling rate while the micropore diameter decreased. The micropores also became more circular when the cooling rate was increased. The pore size and morphology of the P(LA‐co‐CL40) were quite unchanged by varying the cooling rate. All the other conditions being the same, the P(LA‐co‐CL5) foams were better organized than the P(LA‐co‐CL40) foams, and pore orientation was improved at the higher cooling rate. Pore size and morphology also can be controlled by changing the polymer concentration (Cp), as we showed by studying P(LA‐co‐CL5) foams prepared by freeze‐drying solutions in the 1–10 w/v % Cp range. Macropore density, average diameter, and interdistance of P(LA‐co‐CL5) foams increased with Cp, but the micropore characteristics remained almost unchanged no matter the Cp. The reliability of the characterization methods has been discussed, with special attention to mercury intrusion porosimetry, which is used primarily for measurement of pore volume and pore size distribution. However, this technique is reported here as a destructive and unreliable method for the characterization of fragile P(LA‐co‐CL40) foams. This study shows that image analysis and impedance spectroscopy can give reliable information relative to the pore morphology and anisotropy of freeze‐dried foams. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A:199–213, 2003
Piezoelectric and mechanical properties in bovine cornea
Authors: Jayasuriya A. C. , Scheinbeim J. I. , Lubkin V. , Bennett G. , Kramer P.
Published: July 2003

The piezoelectric coefficient (d31) and Young's modulus (E) were investigated as a function of degree of hydration for bovine cornea. The piezoelectric and mechanical responses observed were anisotropic, and d31 decreased, whereas E increased with decreasing the degree of hydration. The anisotropic mechanical and electromechanical properties observed seem to be caused by oriented crystalline collagen fibrils. In addition, the loss of water molecules appears to decrease crystallinity (of the collagen) in the cornea. With dehydration of the cornea, a reduction in crystallinity and changes in hydrogen bonding were observed by wide‐angle X‐ray diffraction and Fourier transform infrared measurements. The decrease of piezoelectricity in cornea during dehydration is most likely caused by the increase in modulus and the loss of order to a nonpiezoelectric phase in the collagen. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 260–265, 2003
Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion
Authors: Keselowsky Benjamin G. , Collard David M. , García Andrés J.
Published: July 2003

Integrin‐mediated cell adhesion to proteins adsorbed onto synthetic surfaces anchors cells and triggers signals that direct cell function. In the case of fibronectin (Fn), adsorption onto substrates of varying properties alters its conformation/structure and its ability to support cell adhesion. In the present study, self‐assembled monolayers (SAMs) of alkanethiols on gold were used as model surfaces to investigate the effects of surface chemistry on Fn adsorption, integrin binding, and cell adhesion. SAMs presenting terminal CH3, OH, COOH, and NH2 functionalities modulated adsorbed Fn conformation as determined through differences in the binding affinities of monoclonal antibodies raised against the central cell‐binding domain (OH > COOH = NH2 > CH3). Binding of α5β1 integrin to adsorbed Fn was controlled by SAM surface chemistry in a manner consistent with antibody binding (OH > COOH = NH2 > CH3), whereas αV integrin binding followed the trend: COOH >> OH = NH2 = CH3, demonstrating α5β1 integrin specificity for Fn adsorbed onto the NH2 and OH SAMs. Cell adhesion strength to Fn‐coated SAMs correlated with α5β1 integrin binding (OH > COOH = NH2 > CH3), and experiments with function‐perturbing antibodies demonstrated that this receptor provides the dominant adhesion mechanism in this cell model. This work establishes an experimental framework to analyze adhesive mechanisms controlling cell‐surface interactions and provides a general strategy of surface‐directed control of adsorbed protein activity to manipulate cell function in biomaterial and biotechnological applications. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 247–259, 2003
Influence of polymeric additives on the biological properties of brushite cements: An experimental study in rabbit
Authors: Flautre B. , Lemaître J. , Maynou C. , Van Landuyt P. , Hardouin P.
Published: July 2003

The resorbability and ability of calcium phosphate hydraulic cements to promote new bone formation was investigated in vivo. The effects of two hydrosoluble polymeric additives (hyaluronic acid, and xanthan gum,) on the biological response of two brushite cement formulations (BHC‐A vs BHC‐B) was investigated. The brushite cements differed in P/Ca (0.71 vs 0.98) and S/Ca (0.10 vs 0.005) atomic ratios and by the presence of calcium sulfate hemihydrate in BHC‐A. Polymer‐free cements were used as controls. Cement specimens were injected in cylindrical bone defects manually drilled in the distal condyle of rabbit femora. The implants were harvested at 12 and 24 weeks after implantation and subjected to quantitative histomorphometry. The study showed a significantly lower resorption rate for cement BHC‐A, which induces the formation of well‐mineralized bone in close apposition to the residual material. In contrast, cement BHC‐B showed a significant increase of bone formation period and the formation of a thick layer of unmineralized osteoid tissue at the bone/residual cement interface. The presence of xanthan gum made the biological response even worse, particularly in the case of cement BHC‐B. The presence of hyaluronic acid has little effect, except for a slight decrease in initial resorption rate, in the case of cement BHC‐A. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 214–223, 2003
Flexible chitin films as potential wound‐dressing materials: Wound model studies
Authors: Yusof Nealda Leila Binte Muhammad , Wee Aileen , Lim Lee Yong , Khor Eugene
Published: July 2003

Chitin films possessing increased flexibility, softness, transparency, and conformability have been prepared. These attributes enable the potential application of chitin films as occlusive, semipermeable film wound dressings similar to commercial products such as Opsite™. The chitin films are generally nonabsorbent, exhibiting a total weight gain of only up to 120–160% in physiological fluid. Dry chitin films transpire water vapor at a rate of about 600 g/m2/24 h, similar to commercial polyurethane‐based film dressings, but rises to 2400 g/m2/24 h, when wet, which is higher than the water vapor transmission rate of intact skin. The chitin films are nontoxic to human skin fibroblasts, maintaining 70–80% cell viability. Wound studies using a rat model showed no signs of allergenicity or the high inflammatory response associated with biodegradable biomaterials. The chitin films displayed accelerated wound‐healing properties. Based on histological examination, wound sites dressed with the chitin films stabilized and healed faster, and appeared stronger than those dressed with Opsite™ and gauze dressings after 7 days of healing. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 224–232, 2003
Preparation of antibacterial silver‐doped silica glass microspheres
Authors: Kawashita Masakazu , Toda Shogo , Kim Hyun‐Min , Kokubo Tadashi , Masuda Noriaki
Published: July 2003

Various types of inorganic substances doped with silver ions have been developed as antibacterial materials, and some have already been commercialized. Colorless and chemically durable materials that slowly release silver ions are, however, still need to be developed. The present authors have previously shown that when a silica glass doped with silver and aluminium ions is prepared using the sol‐gel method, the resultant product is colorless, chemically durable, and slowly releases silver ions into water over a long period. The doped silica glass takes a form of microspheres <1 μm in diameter, it is easily mixed with organic polymers, and the mixture can be formed into a thin film or fine fibers, etc. We report on the preparation of silver doped silica glass microspheres having a diameter =1 μm, using the sol‐gel method. Initially, tetraethoxysilane was partially prehydrolyzed by water in ethanol, and then aluminium triisopropoxide was added to the solution to form SiOAl bonds. Finally, an ammonia solution containing silver nitrate was added to form silica microspheres doped with silver ion together with aluminium ions. The results show monodispersed microspheres 0.4–0.6 μm in diameter were obtained with nominal compositions of Si/Al/Ag = 1/0.01–0.03/0.003–0.03, with a molar ratio of Al/Ag = 1–3.3. The microspheres were colorless, showed a high chemical durability, and slowly released silver ions into water at 37°C. Microspheres with the composition Si/Al/Ag = 1/0.01/0.01 showed excellent antibacterial activity against Escherichia coli. The minimum inhibitory concentration (MIC) of the microspheres was 400, which is less than the MIC value (800) of commercial antibacterial materials. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 266–274, 2003
Exposure of fibrinogen‐adherent platelets to plasma proteins: A new method for studying protein interactions with platelets
Authors: Broberg Marita , Nygren Håkan
Published: July 2003

To understand the effects of mediators in coagulating blood at biomaterial surfaces, it is important to use methods that resemble the normal sequence of events in wound healing around implants. The initial adhesion of platelets from whole blood onto material surfaces is mediated by the fibrinogen receptor glycoprotein IIb/IIIa, as shown in a previous study (Broberg et al., J Lab Clin Med 2002; 139:163–172). In this study, isolated platelets were adhered to fibrinogen and exposed to IgG, von Willebrand factor, or thrombin. The response was detected as the number of adherent platelets, the spreading of platelets, the exposure of CD62P (P‐selectin), and the release of platelet factor 4 (PF4), ADP, and ATP. These results were compared to the response of platelets adhering to surfaces coated with the same proteins. Fibrinogen‐adherent platelets exposed to thrombin generated the significantly highest exposure of CD62P and release of PF4, ADP, and ATP. When platelets were adhered to different protein coatings, von Willebrand factor generated the most CD62P exposure, IgG generated the most PF4 release, and thrombin generated the highest concentration of ADP. These results indicate that protein interactions with platelets may generate different results, depending on the mode of protein exposure. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 403–410, 2003
A preliminary study on the enhancement of the osteointegration of a novel synthetic hydroxyapatite scaffold in vivo
Authors: Damien Elsie , Hing Karin , Saeed Suhur , Revell Peter A.
Published: July 2003

Synthetic hydroxyapatite, a bioactive calcium phosphate, is clinically used as a bone replacement bioceramic because of its similarity in composition to bone mineral, biocompatibility, and osteoconductive nature. The aim of this study was to evaluate the bioactivity of a novel synthetic porous hydroxyapatite (PHA) in vivo in rabbit and to investigate the enhancement of its bioactivity and osteointegration. In the investigation reported here, insulin‐like growth factor‐I (IGF‐I) has been used to enhance the bioactivity of PHA. Cylindrical PHA implants with or without IGF‐I were implanted bilaterally in rabbit femurs. Fluorochrome bone markers were administered at 7‐day intervals. The implants with the attached bone were retrieved at postmortem, 1 and 3 weeks after implantation, for histological and histomorphometric analysis. Undecalcified sections stained with toluidine blue showed new bone formation. Mineralization of the new bone formed in the interface, surrounding trabecular bone, and within the pores of the implants was studied. Lamellar bone mineral apposition rate (MAR) was assessed and compared among treatment groups, sham, PHA alone, and PHA with IGF‐I (500 ng/implant), by fluorochrome label incorporation using UVL microscopy. We report for the first time, that the supplementation of PHA implants with IGF‐I significantly increased new bone formation and MAR (6.58 ± 0.08 μm/day) compared with implantation of PHA alone (4.08 ± 0.05 μm/day) or sham operation (3.11 ± 0.12 μm/day). These results suggest that synthetic PHA might provide a delivery system for bioactive agents to accelerate bone healing in orthopedic procedures. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 241–246, 2003
Engineered cellular response to scaffold architecture in a rabbit trephine defect
Authors: Simon Joshua L. , Roy Tithi Dutta , Parsons J. Russell , Rekow E. Dianne , Thompson Van P. , Kemnitzer John , Ricci John L.
Published: July 2003

Tight control of pore architecture in porous scaffolds for bone repair is critical for a fully elucidated tissue response. Solid freeform fabrication (SFF) enables construction of scaffolds with tightly controlled pore architecture. Four types of porous scaffolds were constructed using SFF and evaluated in an 8‐mm rabbit trephine defect at 8 and 16 weeks (n = 6): a lactide/glycolide (50:50) copolymer scaffold with 20% w/w tri‐calcium phosphate and random porous architecture (Group 1); another identical design made from poly(desaminotyrosyl‐tyrosine ethyl ester carbonate) [poly(DTE carbonate)], a tyrosine‐derived pseudo‐polyamino acid (Group 2); and two poly(DTE carbonate) scaffolds containing 500 μm pores separated by 500‐μm thick walls, one type with solid walls (Group 3), and one type with microporous walls (Group 4). A commercially available coralline scaffold (Interpore) with a 486‐μm average pore size and empty defects were used as controls. There was no significant difference in the overall amount of bone ingrowth in any of the devices, as found by radiographic analysis, but patterns of bone formation matched the morphology of the scaffold. These results suggest that controlled scaffold architecture can be superimposed on biomaterial composition to design and construct scaffolds with improved fill time. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 275–282, 2003
Electrochemical preparation of chitosan/hydroxyapatite composite coatings on titanium substrates
Authors: Redepenning Jody , Venkataraman Guhanand , Chen Jun , Stafford Nathan
Published: July 2003

Composite coatings containing brushite (CaHPO4 · 2H2O) and chitosan were prepared by electrochemical deposition. The brushite/chitosan composites were converted to hydroxyapatite/chitosan composites in aqueous solutions of sodium hydroxide. The coatings ranged from ≈1 to 15% chitosan by weight. Qualitative assessment of the coatings showed adhesion significantly improved over that observed for electrodeposited coatings of pure HA. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 411–416, 2003
Compatibility in vitro of albumin‐heme (O2 carrier) with blood cell components
Authors: Huang Yubin , Komatsu Teruyuki , Nakagawa Akito , Tsuchida Eishun , Kobayashi Shū
Published: July 2003

Recombinant human serum albumin including 2‐[8‐{N‐(2‐methylimidazolyl)}octanoyloxymethyl]‐5,10,15,20‐tetrakis(α,α,α,α‐o‐pivaloylamino)phenylporphinatoiron(II) (albumin‐heme; rHSA‐FeP) is a synthetic hemoprotein that has sufficient capability to reversibly bind and release O2 under physiological conditions (pH 7.3, 37°C) similar to hemoglobin and myoglobin. In order to use this albumin‐based O2 carrier as a new class of red blood cell substitutes, its compatibility with blood cell components carefully was investigated in vitro. After the addition of the rHSA‐FeP solution into whole blood at 10, 20, and 44 vol %, the FeP concentration in the plasma phase remained constant for 6 h at 37°C in each group, and no significant time dependence was observed in the numbers of red blood cells, white blood cells, or platelets. The microscopic observations clearly showed that the shapes of the red blood cells had not been deformed during the measurement period. With respect to the blood coagulation parameters (prothrombin time and activated partial thromboplastin time), the coexistence of rHSA‐FeP had only a negligibly small influence. Also the blood compatibility under dynamic flow conditions was evaluated using a microchannel array flow analyzer. All these results suggest that the albumin‐heme has no effect on the morphology of blood cell components in vitro. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 292–297, 2003
Long‐term craniofacial osteoblast culture on a sodium phosphate and a calcium/sodium phosphate glass
Authors: Gough J. E. , Christian P. , Scotchford C. A. , Jones I. A.
Published: July 2003

The aim of this study was to determine the characteristics of human craniofacial osteoblasts cultured on sodium phosphate glass and calcium–sodium phosphate glass in a long‐term culture of up to 28 days. The characteristics studied were attachment, proliferation, alkaline phosphatase activity, collagen‐1 production, and mineralization. A comparison of the degradation rate, measured by mass loss of the glasses, which are intended for use as a component of a novel degradable composite for craniofacial bone repair, was also performed. It was our hypothesis that the glass would be degradable with a change in degradation rate observed by calcium addition and support osteoblast proliferation and expression of the above characteristics. The inclusion of calcium into the reaction mixture significantly decreased the degradation rate, and it is suggested that the slower degradation is the result of pseudo crosslinking (ionic crosslinks rather than covalent bonding) of the polyphosphate chains by the calcium ions. Therefore, twice as many PO bonds will need to be hydrolyzed for dissolution of the metal phosphate to occur, therefore greatly reducing the rate of hydrolysis. Osteoblasts were able to attach, spread, and proliferate in a manner comparable with the positive control, as shown by analysis of variance. Formation of a collagen‐rich mineralized matrix was also observed. The results presented here suggest that a biocompatible soluble glass has been produced, which has potential to be included in a novel biodegradable craniofacial implant. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 233–240, 2003
Native extracellular matrix coating on Ti surfaces
Authors: Pham Minh T. , Reuther Helfried , Maitz Manfred F.
Published: July 2003

Osteoblast‐like SAOS‐2 cells were allowed to synthesize and assemble their extracellular matrix (ECM) on titanium surfaces. After the selective removal of cells, Ti coated with a native ECM was obtained (ECM‐Ti). The responses of SAOS‐2 cells to ECM−Ti compared with those to peptide sequence RGDS‐ or fibronectin‐immobilized Ti were examined, demonstrating the compositional and structural effects needed to trigger the native cell behavior. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 310–316, 2003
In vitro bioactivity of silicon‐substituted hydroxyapatites
Authors: Balas F. , Pérez‐Pariente J. , Vallet‐Regí M.
Published: July 2003

Silicon‐containing hydroxyapatites were synthesized by the controlled crystallization method. Chemical analysis, N2 adsorption, Hg porosimetry, X‐ray diffraction, scanning electron microscopy‐energy‐dispersive X‐ray spectroscopy, and X‐ray photoelectron spectroscopy (XPS) were used to characterize the hydroxyapatite and to monitor the development of a calcium phosphate layer onto the surface of the substrate immersed in a simulated body fluid, that is, in vitro bioactivity tests. The influence of the silicon content and the nature of the starting calcium and phosphorus sources on the in vitro bioactivity of the resulting materials were studied. A sample of silicocarnotite, whose structure is related to that of hydroxyapatite and contains isolated SiO44− anions that isomorphically substitute some PO43− anions, was prepared and used as reference material for XPS studies. An increase of the unit cell parameters with the Si content was observed, which indicated that SiO44− units are present in lattice positions, replacing some PO43− groups. By using XPS it was possible to assess the presence of monomeric SiO44− units in the surface of apatite samples containing 0.8 wt % of silicon, regardless the nature of the starting raw materials, either Ca(NO3)2/(NH4)2HPO4/Si(OCOCH3)4 or Ca(OH)2/H3PO4/Si(OCOCH3)4. However, an increase of the silicon content up to 1.6 wt % leads to the polymerization of the silicate species at the surface. This technique shows silicon enrichment at the surface of the three samples. The in vitro bioactivity assays showed that the formation of an apatite‐like layer onto the surface of silicon‐containing substrates is strongly enhanced as compared with pure silicon‐free hydroxyapatite. The samples containing monomeric silicate species showed higher in vitro bioactivity than that of silicon‐rich sample containing polymeric silicate species. The use of calcium and phosphate salts as precursors lead to materials with higher bioactivity. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 364–375, 2003
Performance of degradable composite bone repair products made via three‐dimensional fabrication techniques
Authors: Roy Tithi Dutta , Simon Joshua L. , Ricci John L. , Rekow E. Dianne , Thompson Van P. , Parsons J. Russell
Published: July 2003

This study analyzed the in vivo performance of composite degradable bone repair products fabricated using the TheriForm™ process, a solid freeform fabrication (SFF) technique, in a rabbit calvarial defect model at 8 weeks. Scaffolds were composed of polylactic‐co‐glycolic acid (PLGA) polymer with 20% w/w β‐tricalcium phosphate (β‐TCP) ceramic with engineered macroscopic channels, a controlled porosity gradient, and a controlled pore size for promotion of new bone ingrowth. Scaffolds with engineered macroscopic channels and a porosity gradient had higher percentages of new bone area compared to scaffolds without engineered channels. These scaffolds also had higher percentages of new bone area compared to unfilled control defects, suggesting that scaffold material and design combinations could be tailored to facilitate filling of bony defects. This proof‐of‐concept study demonstrated that channel size, porosity, and pore size can be controlled and used to influence new bone formation and calvarial defect healing. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 283–291, 2003
Development of a novel method for synthesis of a polymeric ultrasound contrast agent
Authors: El‐Sherif Dalia M. , Wheatley Margaret A.
Published: July 2003

Medical ultrasound is a highly valuable diagnostic tool, especially when compared with other imaging modalities. It is a noninvasive, real‐time, portable, extremely safe method compared with X‐ray and inexpensive relative to magnetic resonance imaging. However, ultrasound is limited in its ability to distinguish between diseased and normal tissue. This limitation has led to the development of contrast agents. We have produced novel poly (lactic‐co‐glycolic) acid air‐filled microcapsules that work well as ultrasound contrast agents, giving up to 24 and 25 dB enhancement when insonated in the medical imaging range at 5 and 7.5 MHz, respectively. The capsules were fabricated by modifying a double emulsion method to encapsulate camphor in the oil phase and ammonium carbonate in the aqueous phase, and later sublime the encapsulated material, leaving voids capable of being filled with a gas in their place. The role of the surfactant, poly vinyl alcohol, solution temperature, was studied and found to play an important role in the morphology of the capsules, altering their acoustic response. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 347–355, 2003
Preparation, characterization, and in vitro degradation of bioresorbable and bioactive composites based on Bioglass®‐filled polylactide foams
Authors: Maquet V. , Boccaccini A. R. , Pravata L. , Notingher I. , Jérôme R.
Published: July 2003

Highly porous poly(D,L‐lactide)/Bioglass® composites scaffolds were prepared by thermally induced phase separation process of polymer solutions and subsequent solvent sublimation. A series of composite foams with different polymer/Bioglass® weight ratios was prepared to study the influence of Bioglass® content on the foam characteristics such as porous structure, density, and pore volume. The pore volume was decreased from 9.5 to 5.7 cm3/g when the Bioglass® content was increased up to 40 wt %, but the overall pore morphology was not affected very much by changing the polymer/glass composition ratio. The composites foams were then incubated in phosphate‐buffered saline at 37°C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation as an indication of their bioactivity. The addition of Bioglass® to polymer foams increased the water absorption and weight loss as compared with pure polymer foams. However, the polymer molecular weight, determined by size exclusion chromatography, was found to decrease more rapidly and to a larger extent in absence of Bioglass®. This delayed degradation rate in the composite foams was probably caused by the dissolution of alkaline ions from the Bioglass®, resulting in a buffering effect of the incubation medium. After incubation for 7 days, HA was detected by X‐ray diffractometry and Raman spectroscopy and confirmed by environmental scanning electron microscopy and energy‐dispersive X‐ray analysis. The porous composites developed here are promising materials for bone regeneration applications because the formation of HA on the surface of the pore walls should provide good environment for the adhesion and proliferation of osteoblasts and osteoprogenitor cells. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 335–346, 2003
Ultraviolet light‐induced modification of crosslinked hyaluronan gels
Authors: Ramamurthi Anand , Vesely Ivan
Published: July 2003

Hyaluronan (HA) gels (hylans) crosslinked with divinyl sulfone (DVS) are highly biocompatible and can be structurally modified to obtain desired mechanical properties that are attractive for their use as tissue‐engineering scaffolds. However, unmodified hylan gels are not good substrates for cell attachment or infiltration, likely as a result of their smooth surface and the highly anionic nature of HA. This study investigated whether the cell‐adhering characteristics of hylan gels could be enhanced by irradiation with ultraviolet (UV) light, with or without prior dehydration. The attachment and proliferation of neonatal rat smooth muscle cells atop these gels was compared with that on unmodified (control; C) or dehydrated (D) gels. UV‐induced changes to gel structure and chemistry were characterized by confocal and electron microscopy, and fluorphore‐assisted carbohydrate electrophoresis (FACE). Cell attachment was sparse on both unmodified (C) and dehydrated (D) gels. Significantly higher levels of cell attachment were observed on the surface of irradiated (UV) and dehydrated‐irradiated (DUV) gels, likely because of texturing of the gel surface by UV light. In addition, dehydration of gels before UV irradiation created irregular pore‐like structures through which cells appeared to migrate into the interior. FACE assays demonstrated that UV‐irradiation alters the chemistry of HA, causing limited breakdown of HA chains and DVS crosslinks within gel and possibly creating new crosslinks that have not yet been identified. Because the hylan gels are altered structurally and chemically, binding of cells to the material is likely to be more permanent than possible by other approaches, such as coating of cell‐adhesive matrix factors on the gel surface, described previously. The significance of this work is that we have developed a technique for the modification of DVS‐crosslinked HA (hylans) to enhance their performance as a cellular scaffold for tissue‐engineering applications. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 317–329, 2003
Electrostatic spray deposition (ESD) of calcium phosphate coatings
Authors: Leeuwenburgh S. , Wolke J. , Schoonman J. , Jansen J.
Published: July 2003

Using electrostatic spray deposition (ESD), thin calcium phosphate layers were deposited onto commercially pure cp‐Ti substrates. ESD is a thin‐film technique that enables the deposition of inorganic thin films onto metallic substrates using a simple and cheap experimental set‐up. The results show that coating structure and morphology can be tailored by choosing the appropriate combination of deposition parameters. Scanning electron microscopy revealed that various surface morphologies, ranging from dense to very porous coatings, can be obtained. Particularly interesting was a unique reticular coating morphology characterized by a three‐dimensionally interconnected pore network. X‐ray diffraction (XRD) and Fourier transform infrared spectrometry (FTIR) analyses showed that crystalline carbonate apatite coatings were formed after heat treatment of as‐deposited ESD coatings. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 330–334, 2003
Seeding cells into needled felt scaffolds for tissue engineering applications
Authors: Unsworth J. M. , Rose F. R. A. J. , Wright E. , Scotchford C. A. , Shakesheff K. M.
Published: July 2003

Tissue engineering methods are under development that will enable the repair or replacement of a variety of tissues, including articular cartilage and bone. To engineer functional tissue it is necessary that scaffolds initially be seeded with a large number of cells distributed evenly throughout the scaffold structure. It previously has been shown that, compared to static seeding conditions, seeding scaffolds under dynamic conditions facilitates high seeding densities and even distributions of cells (Li et al., Biotechnology Progress 2001;17:935–944). The efficiency of seeding HOSTE85 cells and bovine chondrocytes into needled felt scaffolds following agitation at different speeds was determined. Seeding efficiency was determined using the Hoechst 33258 assay, and cell viability was assessed using the Alamar Blue™ assay. The distribution of cells within the scaffolds was imaged using scanning electron microscopy. It was found that the optimum seeding conditions varied for HOSTE85 cells and bovine chondrocytes, with different agitation speeds leading to different seeding efficiencies, cell viabilities, and distributions of cells within scaffolds. The optimum agitation speeds for seeding a high number of viable cells into scaffolds so that they were arranged evenly were 300 rpm for HOSTE85 cells and 200 rpm for bovine chondrocytes. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 425–431, 2003
Tailored delivery of active keratinocyte growth factor from biodegradable polymer formulations
Authors: Cho Eun Jeong , Tao Zunyu , Tang Ying , Tehan Elizabeth C. , Bright Frank V. , Hicks Wesley L. , Gardella Joseph A. , Hard Robert
Published: July 2003

We report the results of a high throughput screening campaign that is aimed to develop a biodegradable polymer‐based formulation to deliver active keratinocyte growth factor (KGF) and provide a means to tune the KGF delivery rate. A statistical design strategy was used to prepare and screen a series of polymer blends that were composed of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the surfactant sodium bis(ethylhexyl)sulfosuccinate (Aerosol‐OT, AOT). Chloroform was the solvent. Our high throughput screening method used a two‐tiered assessment strategy. At Level 1, we identified “lead” KFG‐loaded formulations that exhibited KGF emission spectra that were the most similar to the native KGF spectrum recorded in buffer. At Level 2, we used steady‐state emission and a homogeneous polarization immunoassay strategy to determine the concentration of total and active KGF, respectively, liberated from the lead formulations during biodegradation. After preparing and screening 2500 formulations, we identified several viable, lead formulations. An analysis of the data showed that the combination of PLA, PGA, and AOT were important to yield a high fraction of active KGF upon release from the formulation; no combination of any two together produced an effect as good as the ternary formulation. The optimum formulations that yielded the highest fraction of active KGF upon release had the following general features: PLA/PGA (w/w) near unity, AOT loading of 100–200 mM, water/AOT mole ratio of 10–20, and a pH between 6 and 8. PLA alone cast from chloroform delivered KGF, but that KGF did not bind to anti‐KGF antibodies (i.e., it was inactive). We can tune the KGF release kinetics by more than two orders of magnitude while maintaining the KGF activity upon liberation from the formulation by adjusting the PLA molecular weight. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 417–424, 2003
Heparin inhibits lipopolysaccharide (LPS) binding to leukocytes and LPS‐induced cytokine production
Authors: Anastase‐Ravion Sylvie , Blondin Catherine , Cholley Béatrice , Haeffner‐Cavaillon Nicole , Castellot John J. , Letourneur Didier
Published: July 2003

The glycosaminoglycan heparin is known to exhibit anti‐inflammatory properties unrelated to its anticoagulant activity. However, in a generalized inflammatory response with implanted or extracorporeal devices, the beneficial effect of heparin coating and/or systemic administration is still unclear as well as the precise mechanisms of action. In the present study, we have first studied the effect of heparin on lipopolysaccharide (LPS)‐induced cytokine production by human blood monocytes. Our results indicated that the production of interleukin‐1α, tumor necrosis factor‐α, and interleukin‐8 was significantly decreased when heparin was simultaneously incubated with Escherichia coli LPS. Because the modulation of heparin on monocyte activation could be mediated by its binding via CD14, the main LPS receptor on monocytes, we then studied the binding of LPS and heparin to leukocytes from human blood and to Chinese hamster ovary cells transfected with the human CD14 gene. The data by flow cytometry showed the binding of biotinylated heparin to leukocytes. Moreover, the experiments performed on leukocytes and on CD14‐positive Chinese hamster ovary cells indicated that heparin inhibited LPS binding. From our results, we conclude that: 1. heparin is an effective inhibitor of LPS‐induced monocyte activation, and 2. heparin inhibits the binding of LPS to cells via a CD14‐independent pathway. This study suggests a potentially important therapeutic application for heparin or heparin analogs to prevent inflammation with biomaterials. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 376–384, 2003
Prevention of polyurethane valve cusp calcification with covalently attached bisphosphonate diethylamino moieties
Authors: Alferiev Ivan , Stachelek Stanley J. , Lu Zhibin , Fu Angela L. , Sellaro Tiffany L. , Connolly Jeanne M. , Bianco Richard W. , Sacks Michael S. , Levy Robert J.
Published: July 2003

Objective: Calcification of polyurethane prosthetic valve leaflets causes a major functional impairment. Previously we showed that polyurethane heart valves modified with covalently linked bisphosphonate groups were resistant to calcification in vivo. However, we also found that the highly polar anionic bisphosphonate groups on the polyurethane surface attracted sodium counter ion adsorption, and thereby increased the elastomer's water absorption to 20% of total weight. In this study we address the increased water absorption by investigating the hypothesis that covalently attaching cationic diethylamino groups to the bisphosphonate‐modified polyurethane will reduce water absorption. Thus we evaluated the mechanical and in vivo anticalcification properties of heart‐valve leaflets composed of this modified polymer. Methods: Diethylamino and bisphosphonate groups (DBP) were appended to the polyurethane Biospan's hard segment using previously published bromoalkylation methodology. Water absorption and biaxial mechanical and uniaxial failure testing were used to determine the mechanical properties of the DBP‐modified polymer. Rat subdermal implants (60 days) and extended (150 days) single pulmonary leaflet replacements in juvenile sheep provided in vivo assessments of the bisphosphonate‐modified polyurethane. Results: The water absorption properties of the DBP‐modified polymers and unmodified polyurethanes were 1.86 and 2.3 %, respectively. Biaxial mechanical tests showed the DBP‐modified polymer was more compliant than the unmodified control material, but all polymeric material had similar uniaxial failure properties. In both rat subdermal and sheep circulatory implants, the DBP‐modified polyurethane resisted calcification, as assessed by scanning electron microscopy, with complete calcification inhibition in prosthetic sheep valve leaflet replacements. Conclusion: DBP polyurethane possesses physical (water absorption) and biomechanical properties comparable to unmodified polyurethane and can resist intrinsic heart‐valve leaflet calcification in blood‐stream implants. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 385–395, 2003
Initiation of translation and cellular localization of Theileria annulata casein kinase IIα: Implication for its role in host cell transformation
Authors: Biermann Reinhild , Schnittger Leonhard , Beyer Doreen , Ahmed Jabbar S.
Published: July 2003

Theileria annulata and T. parva are protozoa that infect bovine leukocytes which leads to subsequent transformation and uncontrolled proliferation of these cells. It has been proposed that the CKIIα subunit of T. parva induces mitogenic pathways of host leukocytes by being exported into the host cell. The evidence for this is the existence of a predicted N‐terminal secretion signal‐like peptide. We tested this hypothesis by analyzing gene structure, translation, and protein localization of the T. annulata CKIIα (TaCKIIα). The determined TaCKIIα‐ORF potentially codes for a 50 kDa protein with an N‐terminal extension including a possible signal sequence not present in CKIIα proteins of non‐Theileria species. However, antisera raised against TaCKIIα recognized a protein of a molecular weight of about 40 kDa and, therefore, inconsistent with this predicted molecular weight. We demonstrate by in vitro transcription/translation that this discrepancy is due to translation from a downstream initiation site omitting the putative N‐terminal signal sequence and thus excluding the notion that the protein product is secreted via the classical secretory pathway. In corroboration immunofluorescence investigations suggest that the TaCKIIα subunit is confined to the parasite schizonts within the host cell. On the basis of the above findings it seems highly unlikely that export via the classical pathway of the parasite CKIIα is the way in which this protein possibly contributes to host cell transformation. J. Cell. Physiol. 196: 444–453, 2003. © 2003 Wiley‐Liss, Inc.
Mechanism of stem cells in the central nervous system
Authors: Johansson Clas B.
Published: July 2003

Injury to the central nervous system (CNS) can result in severe functional impairment. The brain and spinal cord, which constitute the CNS, have been viewed for decades as having a very limited capacity for regeneration. However, over the last several years, the body of evidence supporting the concept of regeneration and continuous renewal of neurons in specific regions of the CNS has increased. This evidence has significantly altered our perception of the CNS and has offered new hope for possible cell therapy strategies to repair lost function. Transplantation of stem cells or the recruitment of endogenous stem cells to repair specific regions of the brain or spinal cord is the next exciting research challenge. However, our understanding of the existing stem cell pool in the adult CNS remains limited. This review will discuss the identification and characterization of CNS stem cells in the adult brain and spinal cord. J. Cell. Physiol. 196: 409–418, 2003. © 2003 Wiley‐Liss, Inc.
Gastrin and EGF synergistically induce cyclooxygenase‐2 expression in Swiss 3T3 fibroblasts that express the CCK2 receptor
Authors: Slice Lee W. , Hodikian Raffi , Zhukova Elena
Published: July 2003

Over‐expression of cyclooxygenase‐2 (COX‐2) has been demonstrated to be tumorigenic in transgenic mice. Chronic treatment with NSAIDs is chemoprotective for colorectal cancer. Gastrin is a growth factor for gastric mucosa and has been shown to promote proliferation of colorectal cells. Recent studies suggest that COX‐2 expression levels could mediate the growth effects of gastrin. Here, we report that gastrin increased PGE2 secretion in Swiss 3T3 cells expressing the CCK2 receptor. Gastrin dose dependently induced COX‐2 protein levels in a time dependent manner. COX‐2 mRNA levels were rapidly induced by a dose dependent increase in gastrin. Prior treatment of the cells with the CCK2 receptor specific antagonist, L365,260, inhibited gastrin‐induced COX‐2 protein and mRNA expression. Pretreatment with L364,714, the CCK1 receptor specific antagonist did not block COX‐2 induction by gastrin. Inhibition of de novo protein synthesis by cycloheximide did not block COX‐2 mRNA induction by gastrin. Also, gastrin‐dependent COX‐2 expression did not require PKC activity, activation of ERK, or transactivation of EGFR. However, co‐stimulation with EGF and gastrin synergistically induced COX‐2 protein and mRNA expression and PGE2 secretion. Measurements of COX‐2 mRNA stability and COX‐2 gene transcription reveal that EGF significantly increased the half‐life of COX‐2 mRNA with only a slight increase in the COX‐2 transcription rate. Conversely, gastrin significantly increased COX‐2 gene transcription rates but did not enhance COX‐2 mRNA stability. J. Cell. Physiol. 196: 454–463, 2003. © 2003 Wiley‐Liss, Inc.
Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting
Authors: Di Marco Eddi , Sessarego Nadia , Zerega Barbara , Cancedda Ranieri , Descalzi Cancedda Fiorella
Published: July 2003

Ex‐FABP, an extracellular fatty acid binding lipocalin, is physiologically expressed by differentiating chicken chondrocytes and myoblasts. Its expression is enhanced after cell treatment with inflammatory stimuli and repressed by anti‐inflammatory agents, behaving as an acute phase protein. Chicken liver fragments in culture show enhanced protein expression after bacterial endotoxin treatment. To investigate the biological role of Ex‐FABP, we stably transfected proliferating chondrocytes with an expression vector carrying antisense oriented Ex‐FABP cDNA. We observed a dramatic loss of cell viability and a strong inhibition of cell proliferation and differentiation. When chondrocytes were transfected with the antisense oriented Ex‐FABP cDNA we observed that Ex‐FABP down‐modulation increased apoptotic cell number. Myoblasts transfected with the same expression vector showed extensive cell death and impaired myotube formation. We suggest that Ex‐FABP acts as a constitutive survival protein and that its expression and activation are fundamental to protect chondrocytes from cell death. J. Cell. Physiol. 196: 464–473, 2003. © 2003 Wiley‐Liss, Inc.
Vasopressin‐induced intracellular redistribution of protein kinase D in intestinal epithelial cells
Authors: Rey Osvaldo , Zhukova Elena , Sinnett‐Smith James , Rozengurt Enrique
Published: July 2003

The spatio‐temporal changes of signaling molecules in response to G protein‐coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady‐state distribution of green fluorescent protein (GFP)‐tagged PKD indicated that in non‐stimulated IEC‐18 cells, GFP‐PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP‐PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)‐mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin‐induced GPCR activation. J. Cell. Physiol. 196: 483–492, 2003. © 2003 Wiley‐Liss, Inc.
Dynamic visualization of expressed gene networks
Authors: Remy Ingrid , Michnick Stephen W.
Published: July 2003

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. While our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact living cells. The experimental strategy is based on protein fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc). In this review, we describe how we go from descriptive to quantitative representations of biochemical networks at an individual to whole genome level and how our approach will lead ultimately to better descriptions of the biochemical machineries that underlie living processes. J. Cell. Physiol. 196: 419–429, 2003. © 2003 Wiley‐Liss, Inc.