bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokeratin 20 and guanylyl cyclase C mRNA is largely present in lymph node and liver specimens of colorectal cancer patients
Authors: Conzelmann Michael , Dieterle Christoph P. , Linnemann Ulrich , Berger Martin R.
Published: September 2003

The aim of our prospective study was to detect circulating epithelial cells (CEC) indicating the presence of disseminated tumor cells (DTC) in tissues affected by lymphatic and hematogenic colorectal cancer metastasis. DTC were tracked in lymph node, liver or bone marrow samples of 245 colorectal cancer patients using 2 independent RT‐PCR assays for cytokeratin 20 (CK20) and guanylylcyclase C (GCC) that demonstrated a sensitivity of 1 colorectal cancer cell in 106 nucleated hematopoietic cells. CK20 mRNA was detected in 79% of lymph nodes, 35% of both liver lobes and 11% of bone marrow samples. GCC mRNA was found in 68% of lymph nodes, 60% of both liver lobes and 6% of bone marrow specimens. Both markers were recorded in 63% of lymph nodes, 45% of at least 1 liver lobe and 1% of bone marrow samples. There was no significant difference when comparing lymph node samples tested positive for both markers in patients with (N1/2; 65%) and without (N0; 56%) nodal involvement. The same was true when comparing the percentages of patients with and without clinically overt distant metastasis who were positive for both markers in at least 1 liver lobe (62% vs. 41%) or in bone marrow (4% vs. 0%). A score denoting the cumulative sum of tests indicating presence of CK20 and GCC mRNA in the liver was significantly related with UICC classification (p = 0.039). However, addition of lymph node results to this score decreased the correlation. The high incidence of clinically inconspicuous lymph node and liver samples tested positive for both markers emphasizes the function of these organs as primary filters for epithelial cells possibly shed from colorectal carcinomas. The potential prognostic significance of these findings warrants verification, especially regarding the importance of CEC or DTC resident in the liver of colorectal cancer patients. © 2003 Wiley‐Liss, Inc.
Interactions between genetic polymorphism of cytochrome P450‐1B1, sulfotransferase 1A1, catechol‐o‐methyltransferase and tobacco exposure in breast cancer risk
Authors: Saintot Monique , Malaveille Christian , Hautefeuille Agnès , Gerber Mariette
Published: September 2003

Genetic polymorphisms of enzymes involved in the metabolism of xenobiotics and estrogens might play a role in breast carcinogenesis related to environmental exposures. In a case‐only study on 282 women with breast cancer, we studied the interaction effects (ORi) between smoking habits and the gene polymorphisms of Cytochrome P450 1B1 (Val432Leu CYP1B1), Phenol‐sulfotransferase 1A1 (Arg213His SULT1A1) and Catechol‐O‐methyltransferase (Val158Met COMT). The smokers carrying the Val CYP1B1 allele associated with a high hydroxylation activity had a higher risk of breast cancer than never smokers with the Leu/Leu genotype (ORi=2.32, 95%CI: 1.00–5.38). Also, the smokers carrying the His SULT1A1 allele associated with a low sulfation activity had a 2‐fold excess risk compared to never smokers carrying Arg/Arg SULT1A1 common genotype (ORi= 2.55, 95%CI: 1.21–5.36). The His SULT1A1 allele increased the risk only in premenopausal patients. The Met COMT allele with a lower methylation activity than Val COMT did not modify the risk among smokers. The excess risk due to joint effect could result from a higher exposure to activated tobacco‐compounds for women homo/heterozygous for the Val CYP1B1 allele. Also, a lower sulfation of the tobacco carcinogens among women with His SULT1A1 could increase exposure to genotoxic compounds. Alternatively, the Val CYP1B1 or His SULT1A1 allele with modified ability to metabolize estrogens could increase the level of genotoxic catechol estrogen (i.e., 4‐hydroxy‐estradiol) among smokers. Our study showed that gene polymorphisms of CYP1B1 and SULT1A1 induce an individual susceptibility to breast cancer among current smokers. © 2003 Wiley‐Liss, Inc.
EGF +61 gene polymorphism and susceptibility to and prognostic markers in cutaneous malignant melanoma
Authors: McCarron Sarah L. , Bateman Adrian C. , Theaker Jeffrey M. , Howell W. Martin
Published: September 2003

CMM is the most serious cutaneous malignancy and is increasing in frequency among most Caucasian populations, where the most important risk factor is exposure to UV light. Relatively little is known of the genetic factors that mediate susceptibility to and prognosis in sporadic CMM, although a number of genes have been implicated. A striking association between EGF polymorphism and Breslow thickness of invasive CMM has been reported. We have sought confirmation of this finding in an independent study of 159 patients and 310 controls using TaqMan fluorescence‐based genotyping for EGF +61. In our study group, there were no significant differences in EGF genotype frequencies between patients and controls nor was EGF genotype associated with tumour growth phase, stage or mitotic count. However, correlation between EGF genotype and Breslow thickness showed a modestly significant increase in frequency of the EGF (G/G) genotype among tumours >3.5 mm thick (30.0% vs. 9.8%, p = 0.03). In summary, in our group, the EGF +61 polymorphism was not a risk factor for CMM susceptibility, but this polymorphism may play a role in disease progression. © 2003 Wiley‐Liss, Inc.
A single nucleotide polymorphism in the transmembrane domain coding region of HER‐2 is associated with development and malignant phenotype of gastric cancer
Authors: Kuraoka Kazuya , Matsumura Shunji , Hamai Yoichi , Nakachi Kei , Imai Kazue , Matsusaki Keisuke , Oue Naohide , Ito Reiko , Nakayama Hirofumi , Yasui Wataru
Published: September 2003

Alterations of the HER‐2 (erbB‐2/neu) proto‐oncogene have been associated with carcinogenesis and poor prognosis of certain cancers. A single nucleotide polymorphism (Ile/Val, A/G) in the transmembrane domain was reported to be associated with a risk of breast cancer. In our study, we examined the association between the HER‐2 polymorphism and gastric carcinoma. The Ile/Ile, Ile/Val and Val/Val genotypes were found in 146 (68.9%), 56 (26.4%) and 10 (4.7%) of 212 gastric cancer patients and in 234 (81.5%), 48 (16.7%) and 5 (1.8%) of 287 control subjects, respectively. The Ile/Val or Val/Val genotype was significantly more frequent in patients than in controls (p = 0.005 and 0.033, respectively). The OR of Val/Val genotype then revealed a significantly enhanced risk of 3.25 (95% CI 1.09–9.70) compared to Ile/Ile genotype; heterozygous Ile/Val genotype showed an intermediate risk of 1.97 (1.27–3.06). In patients, carcinomas of advanced stage were significantly more frequent in patients with Ile/Val or Val/Val genotype than those with Ile/Ile genotype (p < 0.001). The logistic regression analysis for tumor invasion, lymph node metastasis and distant metastasis revealed that lymph node metastasis was most closely associated with the HER‐2 genotype. These results suggest that this nucleotide polymorphism in the transmembrane domain‐coding region of HER‐2 could be associated with development of gastric carcinoma and may serve as a predictor of risk for a malignant phenotype of gastric cancer. The association of HER‐2 genotype with clinicopathologic characteristics of gastric cancer was also suggested, which has to be confirmed with a larger sample size. © 2003 Wiley‐Liss, Inc.
Identification of common or distinct genes related to antitumor activities of a medicinal herb and its major component by oligonucleotide microarray
Authors: Iizuka Norio , Oka Masaaki , Yamamoto Kotaro , Tangoku Akira , Miyamoto Koji , Miyamoto Takanobu , Uchimura Shunji , Hamamoto Yoshihiko , Okita Kiwamu
Published: September 2003

Although the physiological actions of many herbs are gradually being elucidated at the molecular level, it remains unclear how individual components of herbs contribute to their biological activities. In the present study, the antiproliferative activity of Coptidis rhizoma, a medicinal herb, and the major component berberine was investigated in 8 human pancreatic cancer cell lines. Gene expression patterns associated with sensitivities to each agent were analyzed with oligonucleotide arrays that comprised approximately 11,000 genes. We used a tetrazolium dye (MTT) assay to determine ID50 values after the 8 cell lines were exposed to the 2 agents for 72 hr. The ID50 value for berberine was correlated positively with that for C. rhizoma (r=0.725, p=0.0401). C. rhizoma killed tumor cells more effectively than purified berberine when normalized to the level of berberine present in the herb. From the oligonucleotide array data, we selected 20 and 13 genes with strong correlations (r2>0.81) to ID50 values for berberine and C. rhizoma, respectively. Among these 33 genes, the levels of expression of 12 were correlated with the ID50 values of both agents, suggesting that these genes are associated with tumor‐killing activity of berberine in C. rhizoma. Expression of the remaining 21 genes was correlated with the ID50 value of either purified berberine or C. rhizoma. Thus, we identified common and distinct genes responsible for anti‐proliferative activities of purified berberine and C. rhizoma. This strategy may improve our understanding of the actions of herbs with antitumor activities. © 2003 Wiley‐Liss, Inc.
Breast cancer in young women (≤35 years): Genomic aberrations detected by comparative genomic hybridization
Authors: Weber‐Mangal Susanne , Sinn Hans‐Peter , Popp Susanne , Klaes Rüdiger , Emig Robert , Bentz Martin , Mansmann Ulrich , Bastert Gunther , Bartram Claus R. , Jauch Anna
Published: September 2003

Sporadic breast cancer in young women is different from the one in older patients regarding pathological features and aggressiveness of the tumors, but the spectrum of genetic alterations are largely unknown. We used comparative genomic hybridization (CGH) to analyze DNA copy number changes in 88 tumor samples from women ≤35 years of age. Findings were compared to histopathological data including tumor type, grading, lymph nodes and metastasis. Genomic gains clustered to chromosome arms 1q (64.8%), 8q (61.4%), 17q (50.0%), 20q (33.0%), 3q (20.5%), 1p (17.0%), 5p (17.0%) and 15q (17%). Losses were commonly located on 8p (19.3 %), 11q (11.4%), 16q (11.4%), 17p (11.4%) and 18q (10.2%). A comparison with published CGH data from breast carcinomas of similar type and grade showed the following differences: (1) gains were much more frequent than losses, and (2) losses on 8p22–p23 were more prevalent in patients with positive lymph node metastasis (p = 0.02), and Grade III tumors were associated with gains on the long arm of chromosome 8 (p = 0.01). Therefore, alterations in these genomic regions may be responsible for the reduced survival of patients with early onset breast cancer. © 2003 Wiley‐Liss, Inc.
Phase stability after aging and its influence on pin‐on‐disk wear properties of Ce‐TZP/Al2O3 nanocomposite and conventional Y‐TZP
Authors: Tanaka Kenji , Tamura Jiro , Kawanabe Keiichi , Nawa Masahiro , Uchida Masaki , Kokubo Tadashi , Nakamura Takashi
Published: September 2003

Recently zirconia/alumina composites have been examined by many researchers as the new generation of bearing materials in total joint replacements. In this study, the phase stability of a Ce‐TZP/Al2O3 nanocomposite and conventional Y‐TZP after aging, and its influence on wear resistance, were investigated. Very slight phase transformation was observed in both types of ceramics 18 months after the implantation of Ce‐TZP/Al2O3 or Y‐TZP samples into rabbit tibiae. However, Y‐TZP showed marked phase transformation (approximately 80%) after aging in an autoclave (121°C) for 190 h or in physiological saline at 62°C for 18 months, whereas the new composite remained almost resistant to degradation. According to the results of self‐pairing pin‐on‐disk wear tests using ceramic specimens with or without autoclave aging, the wear factor was almost the same between Ce‐TZP/Al2O3 samples with and without aging (6.74 ± 0.36 × 10−8 and 6.04 ± 0.95 × 10−8 mm3/Nm, respectively). In contrast, although non‐aged Y‐TZP had the lowest wear factor (4.88 ± 0.51 × 10−8 mm3/Nm) of all specimens tested, aged Y‐TZP showed 10‐fold greater wear than nonaged Y‐TZP. The present study suggests that Ce‐TZP/Al2O3 nanocomposite has much greater phase stability than Y‐TZP, and that its wear properties are not influenced by aging. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 200–207, 2003
Quantitative evaluation of bacteria adherent to polyelectrolyte HEMA‐based hydrogels
Authors: Berlutti Francesca , Rosso Franco , Bosso Pietro , Giansanti Francesco , Ajello Maria , De Rosa Alfredo , Farina Ernesto , Antonini Giovanni , Valenti Piera
Published: September 2003

The use of adhesive poly(HEMA)‐based hydrogels is standard practice in dental restorative procedures. Microorganisms, which potentially can cause oral pathologies, may colonize these polymers. In the present work, bacterial adhesion to polymers prepared with 2‐hydroxyethyl methacrylate (HEMA) and to different molar ratios of 2‐acrylamido‐2‐methylpropane‐sulfonic acid (AMPS) and/or to 2‐methacryloyloxyethyl‐tri‐methyl‐ammonium chloride (METAC) co‐monomers were tested. A colorimetric assay system that utilizes the Microbo revelation medium (Microbo srl, Rome, Italy) for microbial counts is shown to be capable of counting the number of adherent bacterial cells without removing them from polymer surfaces. In conditions that mimic those present in the oral cavity, similar bacterial adhesion percentages on the same polymer were observed with the different bacteria belonging to both gram‐positive and gram‐negative genera, such as Streptococcus sobrinus and Streptococcus oralis (resident microorganisms in the oral cavity) and Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa (transient microorganisms in the oral cavity). It is determined that the physico‐chemical characteristics of poly(HEMA)‐based hydrogels are the major factors promoting bacterial adhesion, which increased with increasing water content in the swollen polymers, reaching maximal values on the cationic polymers. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 18–25, 2003
Effects of viscoelasticity and time‐dependent plasticity on nanoindentation measurements of human cortical bone
Authors: Fan Zaifeng , Rho Jae‐Young
Published: September 2003

The viscoelastic and time‐dependent plastic effects on the nanoindentation measurement of osteonal lamella in a human cortical bone were investigated. The elastic modulus of osteonal lamella obtained from the quasi‐static technique was strongly affected by the indentation rate and time‐dependent plasticity. The effects of time‐dependent plasticity can be diminished by multiple loading–unloading cycles and a long holding period at maximal load. After minimizing the contribution of time‐dependent plasticity to unloading data, it was surprisingly found that the elastic modulus was proportional to indentation strain rate raised to the 0.06 power, which is similar to conventional test results. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 208–214, 2003
Effects of lithium fluoride and maleic acid on the bioactivity of calcium aluminate cement: Formation of hydroxyapatite in simulated body fluid
Authors: Oh Seung‐Han , Choi Se‐Young , Lee Yong‐Keun , Nam Kim Kyoung , Choi Seong‐Ho
Published: September 2003

To improve the bioactivity of calcium aluminate cement (CAC), which has the potential of restoring defective bone and the joints between artificial prostheses and natural bone, lithium fluoride and maleic acid were added to CAC. Then the bioactivity of the CAC, together with the lithium fluoride and maleic acid, was estimated by examining the hydroxyapatite (HAp) formation on its surface in simulated body fluid (SBF). When 0.5 g of lithium fluoride and 8.75 g of maleic acid were added to 100 g of CAC, LiAl2(OH)7 · 2H2O was formed on the surface of CAC after 1 day of soaking, and HAp was formed after 2 days. The depth of the LiAl2(OH)7 · 2H2O and HAp‐mixed layers after 60 days of immersion was approximately 20 μm. However, after CAC, which contains only 8.75 g of maleic acid per 100 g of CaO · Al2O3, had been soaking for just 30 days, 3CaO · Al2O3 · 6H2O and HAp were detected. These results indicate that lithium fluoride accelerates HAp formation on the surface of CAC in SBF while maleic acid has little influence on HAp formation. The promotion of HAp formation on the surface of CAC in SBF can be explained in terms of the help of an intermediate layer, LiAl2(OH)7 · 2H2O, which contains hydroxyl groups that act as the nuclei of HAp formation and a tremendous dissolution of calcium ions from CAC into the SBF solution within a short induction time. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 104–111, 2003
In vitro bioactivity of S520 glass fibers and initial assessment of osteoblast attachment
Authors: Clupper D. C. , Gough J. E. , Hall M. M. , Clare A. G. , LaCourse W. C. , Hench L. L.
Published: September 2003

Bioactive glass fibers are attractive materials for use as tissue‐engineering scaffolds and as the reinforcing phase for resorbable bioactive composites. The bioactivity of S520 glass fibers (52.0 mol % SiO2, 20.9 Na2O, 7.1 K2O, 18.0 CaO, and 2.0 P2O5) was evaluated in two media, simulated body fluid (SBF) and Dulbecco's modified Eagle's medium (DMEM), for up to 20 days at 37°C. Hydroxyapatite formation was observed on S520 fiber surfaces after 5 h in SBF. After a 20‐day immersion, a continuous hydroxyapatite layer was present on the surface of samples immersed in SBF as well as on those samples immersed in DMEM [fiber surface area to solution volume ratio (SA:V) of 0.10 cm2/mL]. Backscattered electron imaging and EDS analysis revealed that the hydroxyapatite layer formation was more extensive for samples immersed in SBF. Decreasing the SA:V ratio to 0.05 cm2/mL decreased the time required to form a continuous hydroxyapatite surface layer. ICP was used to reveal Si, Ca, and P release profiles in DMEM after the 1st h (15.1, 83.8, and 29.7 ppm, respectively) were similar to those concentrations previously determined to stimulate gene expression in osteoblasts in vitro (16.5, 83.3, and 30.4 ppm, respectively). The tensile strength of the 20‐μm diameter fibers was 925 ± 424 MPa. Primary human osteoblast attachment to the fiber surface was studied by using SEM, and mineralization was studied by using alizarin red staining. Osteoblast dorsal ruffles, cell projections, and lamellipodia were observed, and by 7 days, cells had proliferated to form monolayer areas as shown by SEM. At 14 days, nodule formation was observed, and these nodules stained positive for alizarin red, demonstrating Ca deposition and, therefore mineralization. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 285–294, 2003
In vitro release kinetics of proteins from bioactive foams
Authors: Lenza R. F. S. , Jones J. R. , Vasconcelos W. L. , Hench L. L.
Published: September 2003

This study describes an approach to obtaining 3‐D scaffolds for tissue engineering that allows the incorporation and release of biologically active proteins to stimulate cell function. Laminin was adsorbed on the textured surfaces of binary 70S30C (70 mol % SiO2, 30 mol % CaO) and ternary 58S (60 mol % SiO2, 36 mol % CaO, 4 mol % P2O5) foams. The covalent bonds between the binding sites of the proteins and the ligands on the scaffolds' surfaces did not denaturate the proteins. In vitro studies show that the foams modified with chemical groups and coated with laminin were bioactive, as demonstrated by the formation of a crystalline hydroxy carbonate apatite (HCA) layer formed on the surfaces of the foams upon exposure to simulated body fluid (SBF). The release of proteins from the foams also was investigated. Sustained and controlled release from the scaffolds over a 30‐day period was achieved. Laminin release from the bioactive foams followed the dissolution rate of the material network. These results suggest that bioactive foams have the potential to act as scaffolds for soft‐tissue engineering with a controlled release of proteins that can induce tissue formation or regeneration. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 121–129, 2003
Cell adhesion on polytetrafluoroethylene modified by UV‐irradiation in an ammonia atmosphere
Authors: Heitz J. , Švorčík V. , Bačáková L. , Ročková K. , Ratajová E. , Gumpenberger T. , Bäuerle D. , Dvořánková B. , Kahr H. , Graz I. , Romanin C.
Published: September 2003

We report on the modification of polytetrafluoroethylene (PTFE) by exposure to the ultraviolet (UV) light of a Xe2*‐excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. Typical treatment times were up to 30 min. Subsequently, the samples were grafted with the amino acid alanine from an aqueous solution. The samples were characterized by means of optical transmission spectroscopy, laser‐induced fluorescence and contact‐angle measurements. We studied the adhesion of rat aortic smooth muscle cells (SMC) and mouse fibroblasts (3T3 cells) to the modified polymer samples using an in vitro technique, where the population density and spreadout of adhering cells is determined 24 h after seeding by image analysis. For both cell types the exposure of PTFE to UV‐light in an ammonia atmosphere resulted in a significant increase in the number of adhering cells and in the size of their spreading area. The grafting with alanine enhanced this effect. Additional experiments with human endothelial cells (HEC) also demonstrated improved adhesion to modified PTFE. Thus, PTFE modified by our method appears to be a promising material for fabrication of artificial vascular prostheses and implants or for cultivation of skin substitutes. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 130–137, 2003
Histomorphometric and microhardness assessments of sheep cortical bone surrounding titanium implants with different surface treatments
Authors: Giavaresi G. , Fini M. , Cigada A. , Chiesa R. , Rondelli G. , Rimondini L. , Aldini N. Nicoli , Martini L. , Giardino R.
Published: September 2003

Several factors influence the healing process and the long‐term mechanical stability of cementless fixed implants, such as bone remodeling and mineralization processes. Histomorphometric and bone hardness measurements were taken in implants inserted in sheep femoral cortical bone at different times to compare the in vivo osseointegration of titanium screws (diam.; 3.5 × 7 mm length) with the following surface treatments: machined (Ti‐MA); acid‐etched (Ti‐HF); HA vacuum plasma spray (Ti‐HA); and Ca‐P anodization followed by a hydrothermal treatment (Ti‐AM/HA). Ti‐MA and Ti‐AM/HA implants presented the lowest (Ra = 0.20 ± 0.01 μm) and highest (Ra = 1.97 ± 0.64 μm) significant (p < 0.0005) roughness value, respectively. Bone‐to‐implant contact of Ti‐HF was lower than that of the other surface treatments at both experimetal times (8 weeks: −20%, ns; 12 weeks: −30%, p < 0.01). Significant differences in MAR (mineral apposition rate) were also found between the different experimental times for Ti‐MA (115%, p < 0.01) and Ti‐HF (57%, p < 0.01), demonstrating that bone growth had slowed inside the screw threads of Ti‐HA and Ti‐AM/HA after 12 weeks. No bone microhardness changes in preexisting host bone were found, while Ti‐MA showed the lowest value for the inner thread area at 8 weeks (HV200 μm= 49.8 ± 3.8 HV). These findings confirm that osseointegration may be accelerated by adequate surface roughness and bioactive ceramic coating such as Ca‐P anodization followed by a hydrothermal treatment, which enhance bone interlocking and mineralization. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 112–120, 2003
Matrix metalloproteinase inhibitor within an absorbable coating for vascular applications: Delivery device characterization and the reduction of smooth muscle cell proliferation and migration
Authors: Caldwell Rebecca A. , Vyavahare Narendra , Langan Eugene M. , LaBerge Martine
Published: September 2003

Significant occurrences of arterial restenosis remain a complicating factor of endovascular stent implantation. The incorporation of GM6001, a matrix metalloproteinase inhibitor (MMPI), into a poly(lactide‐co‐glycolide) (PLGA) absorbable coating for 316L stainless steel is proposed as a means to reduce the rate of smooth muscle cell proliferation and migration. Coatings were fabricated using a solvent evaporation technique, and the surfaces were characterized by noncontacting profilometry and scanning electron microscopy. Sufficient degradation of the PLGA determined by gel permeation chromatography occurred to release adequate amounts of the GM6001 from the coating within a 7‐day period. A cumulative GM6001 release at day 42 was determined to be 77.8 ± 1.4% of the actual GM6001 content within the coating. The coating containing the GM6001 reduced the rate of in vitro cell growth of human aortic smooth muscle cell (HASMC) by 30.7 and 37.4% compared to the metallic substrate only after 4 and 7 days, respectively. However, the MMP‐2 activity normalized to cell number was not statistically different between the GM6001 releasing coating and the metal substrate. Using a scrape wound injury assay, the migration of HASMCs was shown to be decreased by 21.4% with GM6001 released from the PLGA coating compared to metallic substrate only. These results suggest that releasing a MMPI from an absorbable coating of a metallic substrate provides a reduction of HASMC proliferation and migration rates, while preserving the overall MMP activity in efforts to retain normal cellular regulation. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1–10, 2003
Immunocytochemical analysis of dentin: A double‐labeling technique
Authors: Breschi L. , Gobbi P. , Lopes M. , Prati C. , Falconi M. , Teti G. , Mazzotti G.
Published: September 2003

Immunocytochemical analysis is a fundamental and selective technique for identifying different molecular components of human dental structure. The hypothesis tested here is that the application of different etching solutions on dentin does not hinder collagen fibrils and proteoglycans from maintaining their immunochemical antigenicity. Human dentin disks were treated with 0.5M of EDTA, citric acid, maleic acid, or phosphoric acid (for 15 or 30 s). A double‐immunolabeling technique was performed to identify, simultaneously, collagen fibrils and chondroitin sulfate. The use of different acids resulted in different degrees of labeling. Maleic and citric acids revealed a diffuse and intense labeling for both collagen fibrils and proteoglycans. The use of phosphoric acid on dentin showed a massive coagulation of the proteoglycans (15 s) or very low labeling (30 s). These data clarify that the use of acids on dentin components is able to modify their antigenicity. Moreover, the double‐labeling immunocytochemical technique allows understanding of the spatial relationships between the collagen fibrils and proteoglycans of the dentin matrix. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 11–17, 2003
Biomimetic peptides that engage specific integrin‐dependent signaling pathways and bind to calcium phosphate surfaces
Authors: Gilbert Michele , Giachelli Cecilia M. , Stayton Patrick S.
Published: September 2003

Many important matrix proteins involved in bone remodeling contain separate domains that orient the protein on hydroxyapatite and interact with target cell receptors, respectively. We have designed two synthetic peptides that mimic the dual activities of these large, complex proteins by binding to calcium phosphate minerals and by engaging integrin‐dependent signaling pathways in osteoblasts. The addition of either PGRGDS from osteopontin or PDGEA from collagen type I to the HAP‐binding domain of statherin (N15 domain) did not alter its α‐helical structure or diminish its affinity for hydroxyapatite. Immobilized N15‐PGRGDS bound MC3T3‐E1 osteoblasts predominantly via the αvβ3 integrin and induced focal adhesion kinase (FAK) phosphorylation at comparable levels to immobilized osteopontin. Immobilized N15‐PDGEA bound MC3T3‐E1 osteoblasts predominantly through the α2β1 integrin and induced similar levels of FAK phosphorylation. Although both peptides induced FAK phosphorylation with similar time courses, only the N15‐PDGEA peptide induced ERK1/2 phosphorylation, showing that these peptides are also capable of engaging integrin‐specific signaling pathways. This peptide system can be used to study adhesion‐dependent control of signaling in the context of the relevant biomineral surface and may also be useful in biomaterial and tissue engineering applications. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 69–77, 2003
Surface mechanical properties of pHEMA contact lenses: Viscoelastic and adhesive property changes on exposure to controlled humidity
Authors: Opdahl Aric , Kim Seong H. , Koffas Telly S. , Marmo Chris , Somorjai Gabor A.
Published: September 2003

The surface mechanical properties of poly(hydroxyethyl)methacrylate (pHEMA)‐based contact lenses were monitored as a function of humidity by atomic force microscopy (AFM). Surface viscoelastic and adhesion values were extracted from AFM force versus distance interaction curves and were found to be strongly dependent on the bulk water content of the lens and on the relative humidity. At low relative humidity, 40–50%, the dehydration rate from the surface is faster than the hydration rate from the bulk, leading to a rigid surface region that has mechanical properties similar to those measured on totally dehydrated lenses. At relative humidity values > 60%, the dehydration rate from the lens surface rapidly decreases, leading to higher surface water content and a softer surface region. The results indicate that, in an ocular environment, although the bulk of the pHEMA contact lens is hydrated, the surface region may be in a transition between a dehydrated glassy state and a hydrated rubbery state. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 350–356, 2003
Ex ova chick chorioallantoic membrane as a novel in vivo model for testing biosensors
Authors: Valdes T. I. , Klueh U. , Kreutzer D. , Moussy F.
Published: September 2003

A major problem with implantable sensors is their short in vivo lifetime, due to strong tissue reactions (i.e., inflammation and fibrosis) caused by the implant and the failure of sensor components. The tissue reactions to the sensor, the biocompatibility of components, and the function of the sensor must be evaluated by using in vivo models. Current methods of in vivo biosensor testing are time‐ and labor‐ intensive and expensive. In addition, the results often vary on the basis of the surgical skills of the investigator. The in ova chorioallantoic membrane (CAM) of the developing chicken embryo was previously developed in our laboratory as a novel in vivo system to test biomaterials. In this new article, we describe a novel approach for testing biosensors in vivo using the ex ova CAM model as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 3 days in ova and then transferred into a petri dish (ex ova) for further incubation at 37°C and 80% humidity. After 1 week of incubation, acetaminophen biosensors, used as model sensors, were placed on top of the CAM and allowed to incorporate for 1 week. Biosensors were then tested for their sensitivity to acetaminophen. CAM venules were injected with 0.2 mL of a 3.6 mM acetaminophen solution. The current produced by the sensor reflected the change in blood acetaminophen levels. Sensors were also assessed by using gross and histological evaluations. We previously reported on the similarity of the tissue response of the CAM with the mammalian models. The low cost, simplicity, and possibility to continuously visualize the sensor test site through a cell culture dish make this animal model particularly attractive for the rapid in vivo screening of biosensors. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 215–223, 2003
Cementum engineering with three‐dimensional polymer scaffolds
Authors: Jin Q.‐M. , Zhao M. , Webb S.A. , Berry J.E. , Somerman M.J. , Giannobile W.V.
Published: September 2003

Cloned cementoblasts (OCCMs), periodontal ligament fibroblasts (SV‐PDLs), and dental follicle (SV‐F) cells obtained from mice were used as a tool to study periodontal tissue engineering. OCCM, SV‐PDL, and SV‐F cells were seeded onto three‐dimensional poly lactic‐co‐glycolic acid (PLGA) scaffolds and cultured with the use of bioreactors or implanted subcutaneously in severe combined immune deficiency (SCID) mice for up to 6 weeks. We explored the behavior of these cells in porous PLGA sponges by cell growth, expression of mineral‐associated genes using reverse transcriptase polymerase chain reaction, and mineralization by histologic analysis in vitro and in vivo. Results indicated that cells attached to PLGA scaffolds under either static or dynamic conditions in vitro. Only OCCM implants, retrieved from both in vitro bioreactors and SCID mice at 3‐and 6‐weeks post–cell implantation exhibited mineral formation. Types I and XII collagens, osteocalcin, and bone sialoprotein genes were detected in all implants retrieved from SCID mice. These results suggest that delivery of selected cells via PLGA scaffolds may serve as a viable approach for promoting periodontal tissue regeneration. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 54–60, 2003
Characterization and analysis of osteopontin‐immobilized poly(2‐hydroxyethyl methacrylate) surfaces
Authors: Martin Stephanie M. , Ganapathy Ramesh , Kim Tae K. , Leach‐Scampavia Deborah , Giachelli Cecilia M. , Ratner Buddy D.
Published: September 2003

A wide array of technologies exist for the characterization and quantification of molecules present at surfaces. We have used several of these experimental and instrumental techniques for the analysis of a novel biomaterial surface. Osteopontin, an extracellular matrix molecule involved in wound‐healing processes, has been chosen as a relevant model protein to immobilize onto poly(2‐hydroxyethyl methacrylate) [poly(HEMA)]. Electron spectroscopy for chemical analysis and time‐of‐flight secondary ion mass spectrometry were used to verify the surface chemistry and the presence of protein. Iodination of osteopontin yielded quantitative data supportive of dose‐dependent immobilization. Enzyme‐linked immunosorbent assay was also used to investigate the presence of osteopontin on poly(HEMA). Finally, the cell adhesive properties of immobilized osteopontin were confirmed by using a bovine aortic endothelial cell adhesion assay. The use of multiple tools to characterize the many facets of a biomaterial surface will undoubtedly improve our understanding of the surface and facilitate the amelioration of in vivo performance. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 334–343, 2003
Action of microparticles of heparin and alginate crosslinked gel when used as injectable artificial matrices to stabilize basic fibroblast growth factor and induce angiogenesis by controlling its release
Authors: Chinen Naofumi , Tanihara Masao , Nakagawa Miyako , Shinozaki Keiko , Yamamoto Eriko , Mizushima Yutaka , Suzuki Yasuo
Published: September 2003

Alginate is an acidic polysaccharide like the glycosaminoglycans and is a candidate for use as an artificial matrix. We developed a novel alginate gel sheet that is crosslinked with heparin (H/A gel sheet) and discovered its properties of releasing biologically active basic fibroblast growth factor (bFGF), a representative member of the heparin‐binding growth factors (HBGFs), for about 1 month in vitro and of inducing angiogenesis in vivo. In the present study, the H/A gel sheet was mechanically broken up to produce easily injectable 50‐ to 200‐μm microparticles of the gel (H/A gel particles), the properties of which were analyzed. The H/A gel particles cumulatively released 2.8 times as much bFGF as the H/A gel sheet, despite both having the same amount of bFGF adsorbed onto their gels. In addition, the bFGF‐adsorbed H/A gel particles released a significant amount of bFGF, which stimulated cellular growth in a culture of human umbilical venous endothelial cells for up to 5 weeks. The subcutaneous injection of the bFGF‐adsorbed H/A gel particles induced the formation of numerous microvessels in the tissue surrounding the gel. These results indicate that the H/A gel particles not only stabilize bFGF by preventing the occurrence of proteolysis or denaturation but also modulate its release from the gel. Because the H/A gel particles can be easily injected into the target tissues, this artificial matrix may be useful for the local delivery of HBGFs in the treatment of ischemic arterial diseases, as well as for regenerating or constructing tissues. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 61–68, 2003
Macroporous poly(N‐isopropylacrylamide) hydrogels with fast response rates and improved protein release properties
Authors: Cheng Si‐Xue , Zhang Jian‐Tao , Zhuo Ren‐Xi
Published: September 2003

A series of temperature‐sensitive poly(N‐isopropylacrylamide) (PNIPA) hydrogels with large pore size and fast response were prepared by carrying out polymerizations in aqueous sodium chloride solutions with different concentrations. In comparison with conventional PNIPA hydrogels, the PNIPA gels thus prepared have remarkably larger swelling ratios below their lower critical solution temperature (LCST), and exhibit much faster response rates as the temperature is raised above their LCST. The improved properties are due to the presence of inorganic salt, NaCl, which leads the phase separation and formation of a heterogeneous porous structure during the polymerizations. The study on the drug release properties shows the macroporous hydrogels exhibit modulated release in response to temperature. The model protein, bovine serum albumin (BSA), can be released completely from the porous hydrogels at the temperature lower than the LCST because the pore size of the hydrogels is larger than the protein molecules. However, the release of BSA from the gels almost stops after a “burst” release in the initial stage at the temperature higher than the LCST because the pores are closed at the high temperature. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 96–103, 2003
Preserving the structure of adsorbed protein films for time‐of‐flight secondary ion mass spectrometry analysis
Authors: Xia Nan , Castner David G.
Published: September 2003

The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein structure. It is desirable to preserve the structure of adsorbed protein films during drying, so UHV analysis could be done in a state that is more representative of proteins' actual structure in the aqueous environment. In this study, two methods, trehalose protection and glutaraldehyde fixation, were explored for their feasibility in preserving adsorbed protein structure for a powerful UHV surface analysis technique, time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). Trehalose protection had shown some promise for ToF‐SIMS analysis in our previous study and was further examined with the model protein fibrinogen in this study. Using the combination of principal component analysis (PCA) and static ToF‐SIMS analysis, we found that trehalose protection could reduce the conformation change of fibrinogen upon drying, and prevent it from unfolding and exposing hydrophobic domains. Moreover, when the adsorbed protein film became more densely packed, the drying‐induced changes in protein structure were reduced. Thus, the protection afforded by trehalose coating was more significant at lower protein surface concentrations. The other method, glutaraldehyde fixation, was used in ToF‐SIMS analysis for the first time. The ϵ‐amino group of lysine was identified as the major reactive group in the protein structure toward glutaraldehyde fixation. Structural differences observed between fibrinogen films that were glutaraldehyde fixed before drying and after drying were similar to those observed between trehalose‐protected and‐unprotected dried fibrinogen films. Glutaraldehyde fixation was found to be a viable, alternative stabilizing method to trehalose protection for ToF‐SIMS analysis. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 179–190, 2003
Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch‐based three‐dimensional scaffolds
Authors: Gomes Manuela E. , Sikavitsas Vassilios I. , Behravesh Esfandiar , Reis Rui L. , Mikos Antonios G.
Published: September 2003

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA‐C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA‐C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6‐well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa‐stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA‐C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three‐dimensional, starch‐based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 87–95, 2003
Microfabricated grooves recapitulate neonatal myocyte connexin43 and N‐cadherin expression and localization
Authors: Motlagh Delara , Hartman Thomas J. , Desai Tejal A. , Russell Brenda
Published: September 2003

Our objective is to alter the surface topography on which cardiac myocytes are grown in culture so that they more closely resemble their in vivo counterparts. Microtextured silicone substrata were made using photolithography and microfabrication techniques and then coated with laminin. Primary cardiac myocytes from newborn rats were plated on microgrooved and nontextured substrata. Myocytes were highly oriented on 5 μm grooves (69.8 ± 2.0%) and significantly different, p < 0.0001, compared with randomly oriented cells grown on nontextured surfaces (2.9 ± 0.95%; n = 19). Cells on shallower, 2 μm, grooves were slightly less well oriented (46.9 ± 4.3%, n = 5, p < 0.001). The lateral spacings of the grooves were altered to examine changes in cell‐to‐cell contact by confocal immunocytochemistry and quantitative protein analysis. Connexin43 and N‐cadherin were distributed around the perimeter of the myocytes plated on 10×5×5 microgrooved surfaces, similar to the localization found in the neonate. Connexin43 expression in cultures on 5 μm deep grooved substrata was equal to the neonatal heart, whereas it differed in nontextured surfaces. We conclude that it is necessary to combine groove depth (5 μm) and lateral ridge dimensions between grooves (5 μm) in order to recapitulate connexin43 and N‐cadherin expression levels and subcellular localization to that of the neonate. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 148–157, 2003
Fourier transform infrared spectroscopy studies of alginate–PLL capsules with varying compositions
Authors: van Hoogmoed Chris G. , Busscher Henk J. , de Vos Paul
Published: September 2003

Microencapsulation of cells is a promising approach to prevention of rejection in the absence of immunosuppression. Clinical application, however, is hampered by insufficient insight into the factors that influence the biocompatibility of the capsules. Capsules prepared of alginates with a high guluronic (G) acid content proved to be more adequate for clinical application since they are more stable, but, unfortunately, they are less biocompatible than capsules prepared of intermediate‐G alginate. In order to get some insight into the physicochemical factors that influence the biocompatibility of capsules for the encapsulation of living cells, the chemical compositions of alginate–Ca beads and alginate–PLL capsules were studied by Fourier transform infrared spectroscopy. We found that during the transition of the alginate–Ca beads to alginate–PLL capsules, Ca connecting the alginate molecules, disappeared at the surface of both high‐G and intermediate‐G alginate–PLL capsules. At the same time, it turned out that high‐G alginate–PLL capsules contained more hydrogen bonding than did intermediate‐G alginate capsules. Thus the well‐known higher stability of high‐G alginate–PLL compared to intermediate‐G alginate–PLL capsules is not caused by a higher degree of binding to Ca of the alginate molecules but rather by the presence of more hydrogen bonds. Another observation was that after the transition from bead to capsule, high‐G alginate–PLL capsules contained 20% more PLL than the intermediate‐G alginate–PLL capsules. Finally, we show that in both high‐G and intermediate‐G alginate–PLL capsules, the PLL exists in the α‐helix, in the antiparallel β‐sheet, and in the random coil conformation. This study shows that FT‐IR allows for successful analyses of the chemical factors essential for understanding differences in the biocompatibility of alginate–PLL capsules. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 172–178, 2003