bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein‐rejecting ability of surface‐bound dextran in end‐on and side‐on configurations: Comparison to PEG
Authors: Österberg E. , Bergström K. , Holmberg K. , Schuman T. P. , Riggs J. A. , Burns N. L. , Van Alstine J. M. , Harris J. M.
Published: December 2004

There is much interest in attaching polyethylene glycol (PEG) and other hydrophilic, neutral polymers to surfaces to reduce the extent of protein and cell adsorption. Interestingly, these same surface‐bound polymers are effective in masking surface charge and reducing electrokinetic effects such as particle electrophoretic mobility, streaming potential, and electroosmosis. It is apparent that similar molecular properties are responsible for both protein and cell rejection and reduction of electrokinetic effects. In this work we compared the fibrinogen‐rejecting ability and the effect on electrophoretic mobility of three polymer coatings bound to polystyrene. The three polymers were side‐bound dextran, end‐bound dextran, and end‐bound PEG. The results of these measurements were used to elucidate the importance of polymer packing density and polymer layer thickness on protein adsorption and reduction of electrokinetic effects. Protein adsorption appears not to be sensitive to polymer layer thickness or the presence of dilute polymer tails in a surface coating, while electrokinetic effects are. Protein adsorption is, however, very sensitive to the availability of exposed surface. Finally, the unique effectiveness of PEG is apparent in this research as in previous studies. © 1995 John Wiley & Sons, Inc.
The resorption of bone‐implanted corals varies with porosity but also with the host reaction
Authors: Roudier M. , Bouchon C. , Rouvillain J. L. , Amédée J. , Bareille R. , Rouais F. , Ch. Fricain J. , Dupuy B. , Kien P. , Jeandot R. , Basse‐Cathalinat B.
Published: December 2004

Three different exoskeletons of coral species Porites astreoides (P), Montastrea annularis (M), and Dichocoenia stokesi (D) were implanted for 2–20 weeks in rabbits. At 2, 4, 8, or 20 weeks, the exoskeletons presented variations in their resorptions depending on the species. To understand the variations in the decreasing speed of the implants despite their similar chemical composition, a study of the surface and architecture of the coral was carried out using scanning electronic microscopy, porosity was evaluated, and growth and differentiation of osteogenic cells cultured in vitro were observed for more than 1 month. At the cellular level, the surface of the implants was identical. Three‐dimensional structures of the implants were variable, but the porosity values [P = 42.7%, M = 40.7%, and D = 17.4%] could not completely account for the differences in the resorbing process of the species. Standard histologic studies performed at 2, 4, 8, and 20 weeks after implantation produced the same pattern with P or M, showing aspects of rapid resorption; however, with D there were images resembling those of a foreign‐body reaction. It seems that when resorption is not quick enough, a foreign body reaction develops which further slows down the process. This work focuses on the importance of porosity when using coral as bone substitute. © 1995 John Wiley & Sons, Inc.
Studies on a new strategy for surface modification of polymeric biomaterials
Authors: Aldenhoff Yvette B. J. , Koole Leo H.
Published: December 2004

A new methodology to improve the hemocompatibility of polyurethane (medical grade Pellethane D‐55) surfaces is reported. The approach is essentially based on a photochemical immobilization reaction. Two new conjugate molecules, compounds 2 and 3, were prepared. They consist of (i) dipyridamole, a well‐known inhibitor of platelet activation, and a vasodilating drug with clinical application, for instance before and during pecutaneous transluminar coronary angioplasty (Dottering); and (ii) an aryl azide, a moiety that exhibits marked photoreactivity. In 2, the dipyridamole unit is directly linked to the aryl azide (via an ester bond), while a short spacer chain separates both units in 3. Upon irradiation of 2 or 3, adsorbed onto the polyurethane foil, the aryl azide is converted into a highly reactive species which reacts with a nucleophilic group on the polymer surface. In this way, the dipyridamole is covalently linked to the polymer. The underlying principle is also used in photoaffinity labeling, a well‐known technique in biochemical studies on enzyme structure and function. From UV extinction experiments it could be deduced that the surfacedensity of immobilized 2 is 4.9 nmol/cm2. The surface density for 3 was 14.6 nmol/cm2. The surfaces were subjected to an in vitro thrombin generation assay. This assay gives a valuable impression about the hemocompatibility of artificial surfaces. These experiments revealed that the clotting times were substantially prolonged as a result of the photoimmobilization of dipyridamole. This was especially the case for immobilized 3. This effect cannot be readily explained. Possibly, the enhanced activity of immobilized 3 is due to the spacer chain. An alternative explanation is that the surface density is larger for 3 than for 2. In addition, the photomodified surfaces were incubated with platelet‐rich blood plasma (37°C, 30 min) and subsequently examined by scanning electron microscopy. The morphology of the blood platelets adhered to the surface also showed that hemocompatibility increased in the order untreated polyurethane < polyurethane with immobilized 2 < polyurethane with immobilized 3. Future work will concentrate on evaluation of the role of the spacer (length, hydrophilicity, etc.), as well as on the possible use of this approach with respect to the construction of biomaterials with improved in vivo biocompatibility, in particular hemocompatibility. © 1995 John Wiley & Sons, Inc.
The influence of surface morphology and wettability on the inflammatory response against poly(L‐lactic acid): A semi‐quantitative study with monoclonal antibodies
Authors: Lam K. H. , Schakenraad J. M. , Groen H. , Esselbrugge H. , Dijkstra P. J. , Feijen J. , Nieuwenhuis P.
Published: December 2004

In this study, the influence of surface morphology and wettability of both degradable and nondegradable polymer films on the inflammatory response after subcutaneous implantation in the rat was investigated. Degradable nonporous, porous, and “combi” (porous with a nonporous layer on one side) poly(L‐lactic acid) (PLLA) films and nondegradable polytetrafluoroethylene (PTFE) and (porous) expanded PTFE (e‐PTFE) were used. Contact angles measurements indicate that PLLA is more hydrophillic than PTFE. Assessment of the inflammatory response was performed after various periods of implantation (up till 180 days), with both conventional light microscopy and immunohistochemistry using monoclonal antibodies (mAbs). The inflammatory response observed initially can largely be considered as part of the wound healing reaction, and up till day 40 the inflammatory response against PLLA was minimally more intense than against PTFE (porous as well as nonporous). From day 40 on, the PLLA films provoke a more intense inflammatory response as compared to the PTFE films. Both porous PLLA and the porous side of the “combi” PLLA film provoke a more intense inflammatory response than nonporous PLLA and the nonporous side of the “combi” PLLA film, respectively. In general, PTFE and e‐PTFE films provoke an inflammatory response which is minimally more intense than the one provoked by the sham operation. Almost no ingrowth of tissue was observed in the porous e‐PTFE films. In contrast, there was abundant tissue ingrowth in and an inflammatory response against porous PLLA. It can be concluded that biodegradable PLLA films provoke a more intense inflammatory response than nondegradable PTFE films. Also, porosity enhances the inflammatory response. However, porosity enhances the inflammatory response only when the wettability of a biomaterial permits cellular ingrowth. © 1995 John Wiley & Sons, Inc.
Activity of enzyme immobilized on silanized Co‐Cr‐Mo
Authors: Puleo D. A.
Published: December 2004

The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co‐Cr‐Mo samples with organic and aqueous solutions of γ‐aminopropyltriethoxysilane (APS) resulted in a concentration‐dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (Gu‐HCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co‐Cr‐Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a miniumum density of approximately 49 NH2 per nm2 of nominal surface area (>0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co‐Cr‐Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity followig treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co‐Cr‐Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity. © 1995 John Wiley & Sons, Inc.
Biological evaluation of the effect of magnetron sputtered Ca/P coatings on osteoblast‐like cells in vitro
Authors: Hulshoff J. E. G. , van Dijk K. , van Der Waerden J. P. C. M. , Wolke J. G. C. , Ginsel L. A. , Jansen J. A.
Published: December 2004

A rat bone marrow cell culture was used to evaluate the osteogenic potential of amorphous and crystalline thin calcium phosphate (Ca/P) coatings. The coatings were deposited on titanium discs using a radiofrequency magnetron sputter procedure. Amorphous and crystalline plasma spray Ca/P coated and noncoated titanium discs served as reference material. The cellular behavior was analyzed with quantitative (attachment and proliferation rates) and qualitative (scanning electron microscopy) techniques. No significant differences were found in cell attachment and proliferation rates between the various materials. Scanning electron microscopy showed extracellular matrix formation after 18 days of culture on amorphous plasma‐sprayed and the two types of magnetron sputtered coatings. Furthermore, no severe degradation of the magnetron sputtered coatings was observed. They even appeared to induce apatite formation. On basis of the results, we conclude that magnetron sputtering appears to be a promising method to manufacture bioactive ceramic coatings. © 1995 John Wiley & Sons, Inc.
The use of xenogeneic small intestinal submucosa as a biomaterial for Achille's tendon repair in a dog model
Authors: Badylak Stephen F. , Tullius Robert , Kokini Klod , Shelbourne K. Donald , Klootwyk Thomas , Voytik Sherry L. , Kraine Meredith R. , Simmons Cassandra
Published: December 2004

A study was conducted to evaluate the tissue response to a xenogeneic biomaterial when this material was used to repair an experimentally induced Achilles tendon defect in the dog. Twenty dogs had a 1.5 cm segmental defect of the Achilles tendon created surgically which was then reparired with acellular connective tissue derived from porcine small intestinal submucosa (SIS). The animals were sacrificed at 1, 2, 4, 8, 12, 16, 24, and 48 weeks and the neotendons examined for uniaxial longitudinal tensile strength, morphologic appearance, hydroxyproline (collagen) content, and disappearance of the originally implanted SIS material over time. The contralateral normal Achilles tendons served as controls as did four additional dogs that had a 1.5 cm segmental Achilles tendon defect created surgically without subsequent surgical repair with SIS. Results showed the SIS remodeled neotendons to be stronger than the musculotendinous origin or the boney insertion (>1000 N) by 12 weeks after surgery and to consist of organized collagen‐rich connective tissue similar to the contralateral normal tendons. The four dogs in which no SIS was implanted showed inferior strength at the comparable time points of 4, 8, 12, and 16 weeks. Immunohistochemical studies suggest that the SIS biomaterial becomes degraded within the first eight weeks and serves as a temporary scaffold around which the body deposits appropriate and organized connective tissue. SIS is a promising biomaterial worthy of further investigation for orthopedic soft tissue applications. © 1995 John Wiley & Sons, Inc.
A transmission electron microscopy examination of the interface between osteoblasts and metal biomaterials
Authors: Garvey B. T. , Bizios R.
Published: December 2004

Transmission electron microscopy was used to examine the interface between metal implant materials and bone cells. Specifically, neonatal rat calvaria osteoblasts were cultured on CoCrMo alloy and on 316L stainless steel discs (mechanically polished to a 0.3 μm finish) in Dulbecco's Modified Eagle Medium (supplemented with 10% fetal bovine serum, 50 μg/mL ascorbic acid, and 10 mM ß‐glycerophosphate) under standard, sterile, cell culture conditions for 14 to 28 days. At the end of the prescribed time periods, the cells were fixed and embedded in resin before removing the metal substrates using an electrolytic dissolution technique and a 7% NaCl solution. Transmission electron microscopic examination of stained, ultrathin sections of the biological samples revealed an intact interface with microscopic details characteristic to the cell line and similar to those reported in the literature for animal and explant studies. The osteoblasts exhibited continous contact and intimate apposition to both the CoCrMo and stainless steel substrate surfaces and grew in multilayered structures; an electron dense layer (composed of mucopolysaccharides and proteins) was observed at the surface of both substrates; collagen fibrils and mineralized foci were observed in the extracellular matrix interspersed among the multilayered osteoblasts. © 1995 John Wiley & Sons, Inc.
Modulation of adhesive properties of DEAE‐dextran with laminin
Authors: Varani James , Fligiel Suzanne E. G. , Inman Dennis R. , Beals Ted F. , Hillegas William J.
Published: December 2004

Human squamous epithelial cells produce lower amounts of laminin and fibronectin when cultured on DEAE‐dextran than when cultured on gelatin‐coated polystyrene (Biotechnol. Bioeng., 33:1235). The epithelial cells also spread much more slowly on DEAE‐dextran than they do on gelatincoated polystyrene. To determine if the low level of matrix production by cells grown on DEAE‐dextran contributed to the slowness of cell spreading on this substrate, microcarriers made from DEAE‐dextran were treated with exogenous laminin (10 μg/cm2 of surface area) and then examined for ability to support cell adhesion. Squamous epithelial cells spread as rapidly on the laminin‐treated DEAE‐dextran as they did on gelatin‐coated polystyrene (much more rapidly than on untreated DEAE‐dextran). This indicates (1) that laminin can bind to DEAE‐dextran in a fashion that is biologically usable by anchorage‐dependent cells, and (2) that when laminin is bound to DEAE‐dextran, the failure of squamous epithelial cells to rapidly spread is overcome. These data support the hypothesis that failure of the cells to synthesize an intact extracellular matrix on DEAE‐dextran is responsible, at least in part, for the slowness with which cells spread on this substrate. © 1995 John Wiley & Sons, Inc.
Ultrastructural changes in cultured human periodontal ligament cells exposed to dental materials
Authors: Adams A. M. , Soames J. V. , Searle R. F.
Published: December 2004

The cytopathic effects of a range of dental restorative cements were examined by electron microscopy using an in vitro model with cultured test cells derived from human periodontal ligament. Monolayers were fixed and processed in situ after 2, 24, and 48 h exposure. Many cells showed lysis after two hours exposure consistent with immediate acute injury. Cultures subsequently recovered, reflecting different susceptibilities of cells to injury, and at later stages showed distinct patterns of cell damage in response to different restorative materials. These were related primarily to either cytoplasmic or nuclear damage and to changes resembling apoptosis. © 1995 John Wiley & Sons, Inc.
Contact activation of the plasma coagulation cascade. I. Procoagulant surface chemistry and energy
Authors: Vogler Erwin A. , Graper Jane C. , Harper Garry R. , Sugg Harry W. , Lander Lorraine M. , Brittain William J.
Published: December 2004

Contact activation of the intrinsic pathway of porcine blood plasma coagulation is shown to be a steep exponential‐like function of procoagulant surface energy, with low activation observed for poorly water‐wettable surfaces and very high activation for fully water‐wettable surfaces. Test procoagulants studied were a system of oxidized polystyrene films with varying wettability (surface energy) and glass discs bearing close‐packed self‐assembled silane monolayers (SAMs) with well‐defined chemistry consisting of 12 different terminating chemical functionalities. A monotonic trend of increasing coagulation activation with increasing water wettability was observed for the oxidized polystyrene system whereas results with SAM procoagulants suggested a level of chemical specificity over and above the surface energy trend. In particular, it was noted that coagulation activation by SAMs terminated with CO2H was much higher than anticipated based on surface wettability whereas NH3⊕‐terminated SAMs exhibited very low procoagulant activity. SAMs terminated in (CH2)2(CF2)7CF3 behaved as anticipated based on surface energy with very low procoagulant activity and did not exhibit special properties sometimes attributed to perfluorinated compounds. Quantitative ranking of the inherent coagulation activation properties of procoagulant surfaces was obtained by application of a straightforward phenomenological model expressed in a closed‐form mathematical equation relating coagulation time to procoagulant surface area. Fit of the model with a single adjustable parameter to experimental measurements of porcine platelet‐poor plasma coagulation time was very good, implying that assertions and simplifications of the model adequately simulated reality. Two important propositions of the model were that (1) the number of putative “activating sites” scaled linearly with procoagulant surface area, and (2) contact activation of the plasma coagulation cascade was catalytic in the sense that these activating sites were not consumed or “poisoned” by irreversible or slowly reversible protein adsorption during coagulation. An extension of the coagulation model proposed that procoagulant activation properties scale exponentially with the surface density of polar (acid‐base) sites, which, in turn, was related to procoagulant wettability. © 1995 John Wiley & Sons, Inc.
Photochemical surface derivatization of a peptide containing Arg–Gly–Asp (RGD)
Authors: Sugawara Takashi , Matsuda Takehisa
Published: December 2004

A photoreactive octapeptide (Gly–Gly–Gly–Arg–Gly–Asp–Ser–Pro), into which the cell adhesive tripeptidyl sequence Arg–Gly–Asp (RGD) was incorporated, was derivatized with 4‐azidobenzoyloxysuccinimide at its N‐terminal. Photochemical surface immobilization of the RGD peptide on poly (vinyl alcohol) (PVA) was performed by adsorption and subsequent ultraviolet (UV) irradiation and was evidenced by electron spectra for chemical analysis (ESCA) measurements. Bovine endothelial cells (ECs) adhered and spread well on the RGD peptide‐derivatized surfaces in a biologically specific manner. The use of a photomask during UV irradiation enabled preparation of a two‐dimensional tissue with micron‐order precision. Thus surface immobilization of a photoreactive, cell‐adhesive peptide easily can create a cell attachment surface at a desired portion or region of a polymer surface or device. © 1995 John Wiley & Sons, Inc.
Comparison of the release of growth hormone from hydroxyapatite, heat‐treated hydroxyapatite, and fluoroapatite coatings on titanium
Authors: Downes S. , Clifford C. J. , Scotchford C. , Klein C. P. A. T.
Published: December 2004

Hydroxyapatite (HA), heat‐treated hydroxyapatite (HAH), and fluorapatite (FA) coatings on titanium were loaded with human growth hormone (GH), and the subsequent release was monitored in vitro. The amount of GH released was significantly increased from the HA coating that had received a post‐plasma‐spraying heat treatment prior to incorporation of the growth hormone. Scanning electron microscopy was used to assess the surfaces of the ceramic coatings prior and postincubation with GH. Surface changes were observed on the HA and HAH coatings but not on the FA coatings after incubation with GH. Osteoblast‐like cells were grown on the coatings and maintained in culture. Scanning electron microscopy was used to study the morphology of the cells and the interaction of the cells with the ceramic coatings. We used thymidine uptake and DNA content to determine the relative rates of cell division on the different coatings; the optimum rate of cell proliferation was observed on the HAH coating loaded with 0.1 IU/mL GH. © 1995 John Wiley & Sons, Inc.
Bone‐bonding behavior under load‐bearing conditions of an alumina ceramic implant incorporating beads coated with glass–ceramic containing apatite and wollastonite
Authors: Li Zhong‐Lian , Kitsugi Toshiaki , Yamamuro Takao , Chang Yong‐Sun , Senaha Yoshinaga , Takagi Haruki , Nakamura Takashi , Oka Masanori
Published: December 2004

Alumina ceramic with a porous surface coated with glass‐ceramic containing apatite and wollastonite (AW‐GC) was implanted in a state of press‐fit under loas‐bearing conditions in the femoral condylus of the mongrel dog and compared with a non‐glass‐ceramic‐coated alumina ceramic. A trapezoid alumina ceramic implant (7 × 10 × 5 mm) with a lateral recess (0.9 mm deep) coated with alumina ceramic beads (mean diameter, 750 μm) in a single layer was prepared. The alumina ceramic beads were bonded to the alumina ceramic substratum using an identical alumina binder. The thickness of coating was 10–50 μm (mean, 30 μm). The surface of the beads and the substratum of the alumina implant were coated with AW‐GC. A pull‐out test and histologic examination were performed at 4, 8, and 24 weeks after implantation. The interfacial shear load was significantly increased from 8 to 24 weeks in both groups. The shear load of the glass‐ceramic‐coated implant was significantly greater than that of the noncoated implant at every stage. The interface shear load of the noncoated implant was 12.13 ± 2.76 kg at 4 weeks, 13.92 ± 4.18 kg at 8 weeks, and 24.17 ± 5.17 kg at 24 weeks after implantation. The interface shear load of the glass‐ceramic‐coated implant was 17.96 ± 2.81 kg at 4 weeks, 24.92 ± 9.87 kg at 8 weeks, and 34.83 ± 4.12 kg at 24 weeks after implantation. Histologic examination showed more ingrown bone tissue in the glass‐ceramic‐coated implants. It is suggested that AW‐GC stimulated the bone ingrowth. The bonding behavior of bioinert alumina ceramics is therefore improved by a coating of bioactive glass‐ceramic. © 1995 John Wiley & Sons, Inc.
Cellular responses to chemical and morphologic aspects of biomaterial surfaces. I. A novel in vitro model system
Authors: Chesmel K. D. , Black J.
Published: December 2004

The clinical success of any implant is directly dependent upon the cellular behavior in the immediate vicinity of the interface established between the host tissue and the biomaterial(s) used to fabricate the device. All biomaterials have morphologic, chemical, and electrical surface characteristics that influence the cellular response to the implant. Quantitative measurement of specific aspects of this local host response to different but well‐characterized biomaterial surfaces provides a crucial link in the understanding of the overall phenomenon of implant biocompatibility. A system has been devised for in vitro examination of responses of cells to controlled but independent changes in both the chemistry and morphology of polystyrene (PS) tissue culture surfaces. Micromachined silicon wafers were used as templates to solvent‐cast PS replicas [using 0, 1, or 2 wt % styrene (S) monomer additions] with either none, 0.5‐ or 5.0‐μm‐deep surface grooves arranged in a radial array. When all possible morphologies were combined with all possible polymers, nine model biomaterial surfaces (MBSs) were produced. The chemical characteristics of the MBSs were determined using electron spectroscopy for chemical analysis, secondary ion mass spectroscopy, and contact angle techniques and were found to be distinct. The types and amount of proteins that adsorb onto these surfaces from serum containing media were examined and found to consist of multiple molecular layers of relatively uniform composition. Self‐contained tissue culture vessels formed from the MBSs were capable of supporting the growth of confluent cultures of rat calvarial cells. The model biomaterial system described here can be used to examine how simultaneous stimuli resulting from the chemical and morphological characteristics of a test material may influence biologic responses. Such multifactorial biocompatibility research is needed to properly document material‐host interactions. © 1995 John Wiley & Sons, Inc.
Local delivery of indomethacin by a polyorthoester inhibits resossification of experimental bone defects
Authors: Solheim Eirik , Pinholt Else Mari , Andersen Rune , Bang Gisle , Sudmann Einar
Published: December 2004

Inhibition of orthotopic reossification after surgical removal of bone is sometimes indicated and may be accomplished by implantation of interpositional materials or by systemic administration of indomethacin. However, implantation of nonresorbable foreign material may induce a chronic inflammation and predispose to infections; and systemic administration of indomethancin may induce systemic adverse effects. We studied the effect of local delivery of indomethacin by a bioerodible polyorthoester on the reossification of segmental defects of the radius in rats. We divided 45 Wistar rats into three groups, A–C. A 3.5 mm‐long middiaphyseal osteoperiosteal resection of the right radius was made in each rat. The defect was filled with 15 mg of polyorthoester with 5% indomethacin in group A and 15 mg of polyorthoester without drug in group B. No material was implanted in the defects in the group C rats. The rats were killed 50 days postoperatively. The mean area of the residual defects were greater in the defects with the polyorthoester with 5% indomethacin compared with defects with polyorthoester without drug or without implant as judged by computer‐assisted area measurements on radiographs. By light microscopy, no inflammation was seen and only traces of the polyorthoester could be dected in the defects filled with the polyorthoester with or without indomethacin. The results of this study suggest that the polyorthoester may be used as a bioerodible system for local delivery of indomethacin to inhibit reossification of skeletal defects without tissue reaction, unabsorbed carrier, or systemic effects. © 1995 John Wiley & Sons, Inc.
Polysulphone inhibits final differentiation steps of osteogenesis in vitro
Authors: van Loon Jack J. W. A. , Bierkens Johan , Maes Jef , Schoeters Greet E. R. , Ooms Daniella , Doulabi Behrouz Zandieh , Veldhuijzen J. Paul
Published: December 2004

Biocompatibility is an important factor in the development of orthopedic implants as well as in the development of new tissue culture devices. Polysulphone has been used for orthopedic implants because of its mechanical properties, ease of sterilization, molding capacity, and biocompatibility. Therefore, polysulphone has been chosen as the prime material for the construction of tissue culture devices to be used for the cultivation of osteogenic cells (preosteoblastlike MN7 cells and primary bone marrow fragments), as well as complete fetal long bone explants under space flight conditions. Whereas polysulphone did not interfere with the proliferation in early stages of bone‐forming cells, we show that leachable factors within the polysulphone polymer prevented the final steps of matrix formation as measured by collagen synthesis and matrix mineralization. These data argue against polysulphone as a material for orthopedic implants. © 1995 John Wiley & Sons, Inc.
Integral Membrane Glycoproteins Related to Cell–Substratum Adhesion in Mammalian Cells
Authors: Damsky Caroline H. , Knudsen Karen A. , Buck Clayton A.
Published: December 2004

Broad spectrum antisera have been raised against surface membrane‐derived material from baby hamster kidney cells and mouse mammary tumor epithelial cells. These antisera disrupt cell–substratum adhesion in their respective cell types. Using an antibody neutralization (blocking) assay, adhesion‐related glycoproteins have been isolated from non‐ionic detergent extracts of each cell type. The purified material in each case consisted of a restricted population of glycoproteins of approximately 120,000–160,000 Mr. Purified material from each system blocked the disruption of adhesion induced by the heterologous antiserum on either cell type. The antisera were capable of disrupting cell–substratum adhesion of a large number of cell types and species sources. In addition, antibody blocking activity could be detected from partially purified extracts of several adult hamster cell types and a variety‐of cultured cell types. Thus, in addition to having similar substratum–associated glycoproteins (eg, fibronectin) and cytoskeleton‐associated proteins (eg, α‐actinin and vinculin) cells from different species and tissue sources appear to have a relatively conserved class of integral membrane glycoproteins involved in cell substratum–adhesion.
Growth of Human T Lymphocyte Colonies From Whole Blood: Culture Requirements and Applications
Authors: Knox S.J. , Wilson F.D. , Greenberg B.R. , Shifrine M.
Published: December 2004

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient‐enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low‐level radiation effects on lymphocytes than widely used tests that measure 3H‐thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalties in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.
Differential Reactivation of Zinc‐Mediated Metallothionein Induction in Ultraviolet‐Irradiated Normal and Repair‐Deficient Human Cells
Authors: Hildebrand C.E. , Strniste G.F.
Published: December 2004

The ubiquitous, low‐molecular‐weight, thiol‐rich, metal‐binding protein, metallothionein (MT), can be induced in cultured normal human fibroblasts (NF) and xeroderma pigmentosum (XP) cells by exposure to ZnCl2. Both NF and XP cells tolerate up to 200 μM ZnCl2 in the growth medium. Upon addition of ZnCl2 (200 μM) to monolayer cultures, both NF and XP cells showed similar kinetics for the induction of MT synthesis: Within 7 hours the MT synthesis rate rose from a low, marginally detectable rate to a maximal rate at least 50‐fold greater than the basal rate. The induction of MT synthesis in both cell types was inhibited by actinomycin D (5 μg/ml), indicating that the induction process is controlled at the level of transcription. Exposure of NF or XP cells to far ultraviolet light (UV) followed by induction with ZnCl2 resulted in a UV dose‐dependent decrease in the maximal rate of MT synthesis measured 8.5 hours postirradiation. The UV sensitivity of the MT induction was greater in XP cells than in NF cells. However, considerations of the differential repair capacities of NF and XP cells superimposed upon the kinetics of MT induction were invoked to explain the apparent differential UV sensitivity of MT induction. Liquid holding recovery experiments showed that NF cells possess the capacity to reactivate this inducible gene function rapidly while XP cells arc deficient in the reactivation capacity. These results are discussed in the context of both UV transcriptional mapping of this inducible gene function and development of techniques for measuring repair of transcription‐blocking lesions.
Carcinogen‐Mediated Amplification of Specific DNA Sequences
Authors: Lavi Sara
Published: December 2004

A model experimental system based on SV40‐transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen‐mediated amplification of SV40 DNA sequences is regulated by the viral “A” gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.
Expression of Adhesion‐Related Membrane Components in Adherent Versus Nonadherent Hamster Melanoma Cells
Authors: Knudsen Karen A. , Damsky Caroline H. , Buck Clayton A.
Published: December 2004

The existence of integral membrane components that are involved in cell–substratum adhesion has been postulated. Using an immunochemical approach developed in this laboratory, we provide further evidence for the role in cell–substratum adhesion of integral membrane glycoproteins within a molecular weight region of 120,000–140,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of material enriched approximately 100‐fold in adhesion‐related components revealed the 120,000–140,000 Mr glycoproteins in an adherent hamster melanoma cell line. These glycoproteins are greatly reduced in a non‐adherent variant. Induction of adhesion in these cells by exposure to BudR is accompanied by re‐expression of the surface adhesion antigens.
Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation‐Defective Mutants of Dictyostelium discoideum
Authors: Armant D. Randall , Berger Edward A.
Published: December 2004

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two‐stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with 125I‐Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread‐sorption of the antiserum with discoidin‐Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified 125I‐discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells.

These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation‐defective mutants. Strain EB‐32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB‐18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.
Evidence for the Functional Association of Enzyme I and HPr of the Phosphoenolpyruvate‐Sugar Phosphotransferase System With the Membrane in Sealed Vesicles of Escherichia coli
Authors: Saier Milton H. , Cox David F. , Feucht Brigette U. , Novotny Michael J.
Published: December 2004

Several independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate‐sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate‐dependent sugar phosphorylation was catalyzed by this membrane‐bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS‐enzyme complex was stabilized by Mg++ and phosphoenolpyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.
Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity
Authors: Nato Farida , Bourrillon Roland
Published: December 2004

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells.

The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO4 followed by reduction with NaB3H4. Surface‐labeled glycoproteins were released by short incubations with TPCK‐trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II2, was then purified by chromatography on DEAE‐cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS‐polyacrylamide gels, isoelectric focusing, rechromatography on DEAE‐cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons.

The location of GP II2on the cell surface was confirmed by the fact that it could be labeled metabolically with, D‐(3H) glucosamine and externally through the nonpenetrating periodate‐NaB3H4 system.

GP II2could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II2could be an integral protein.

Analysis of the carbohydrate composition of GP II2 revealed galactose, N‐acetylglucosamine, N‐acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O‐ and N‐linked to the polypeptide chain, most of them being O‐linked.

Finally, GP II2has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.
Responses of Preneoplastic Epidermal Cells to Tumor Promoters and Growth Factors: Use of Promoter‐Resistant Variants for Mechanism Studies
Authors: Colburn Nancy H. , Wendel Edmund , Srinivas Leela
Published: December 2004

The JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second‐stage but not first‐stage promoters, and is inhibited by inhibitors of second‐stage not first‐stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen‐resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen‐resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation.
Differences in Metabolism of Chemical Carcinogens in Cultured Human Epithelial Tissues and Cells
Authors: Harris Curtis C. , Trump Benjamin F. , Grafstrom Roland , Autrup Herman
Published: December 2004

The metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen‐DNA adducts are generally qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in out‐bred animal species, including humans. These interindividual differences in amounts of carcinogen‐DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10‐to 150‐fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.
Changes in the Expression of Membrane Antigens During the Differentiation of Chicken Erythroblasts
Authors: Hayman Michael J. , Beug Hartmut , Savin Keith W.
Published: December 2004

Chicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a “block” in differentiation, in that when erythroblasts are transformed by a temperature‐sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid‐specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid‐specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.
Structural Characteristics of the Short‐Tail Fibers of T4 Bacteriophage
Authors: Zorzopulos Jorge , DeLong Sara , Chapman Virginia , Kozloff Lloyd M.
Published: December 2004

The characteristics of pure preparations of short‐tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43‐nm fibers were the most abundant structure. During the process of purification of the short‐tail fibers, the formation of aggregates was observed each time the material containing the short‐tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short‐tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short‐tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well‐defined bundles. The amino acid composition of the highly purified short‐tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short‐tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short‐tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.
The Role of Regulatory Components From Resident T Lymphocytes in Polyclonal B Cell Activation
Authors: Goodman Michael G. , Weigle William O.
Published: December 2004

Resident T lymphocytes have been found to exert helper and suppressor regulatory influences with regard to polyclonal activation of murine splenic B lymphocytes elicited by lipopolysaccharide. In the normal adult spleen, only T cell helper influences are exercised over polyclonal B cell activation. This activity is a property of Lyt 1+2− T cells and does not appear to be subject to MHC restriction. Suppressive influence evidently is either latent or it exists at such a low level that its effects are difficult to detect. No regulatory activity can be recovered from the supernatants of T cells, cultured either with or without LPS. However, suppressor T cell function may be evoked by activating splenic T cells with Concanavalin A or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these unstimulated cells when intact. The soluble fraction of resident splenic T cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt 1+2− T cells, whereas suppressor activity is generated from Lyt 1−2+ T cells. The suppressive activity of T cell sonicates is not restricted by the MHC gene complex. Helper and suppressor activities contained in splenic T cell sonicates were separated by gel chromatography; the suppressive activity was found to elute with a molecular weight between 68,000 and 84,000 daltons, and the helper activity eluted with a molecular weight between 15,000 and 23,000 daltons. The data indicate that helper and suppressor activities are distinct molecular entities derived from distinct splenic T lymphocyte subpopulations. The possibility that these molecules are precursors to or components of antigen‐specific or nonspecific helper and suppressor factors described in the literature is discussed.