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1.  Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces 
To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces.
Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot.
There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface.
These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs.
PMCID: PMC3774950  PMID: 24049577
Transmucosal abutment; Focal adhesion linker proteins; Gingival fibroblast; Adhesion
2.  Effect of growth factors (BMP-4/7 & bFGF) on proliferation & osteogenic differentiation of bone marrow stromal cells 
Background & objectives:
BMP (bone morphogenetic protein)-4/7 and bFGF (basic fibroblast growth factor) significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells), respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro.
BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A) 80 ng/ml BMP-4/7; (B) 80 ng/ml bFGF; (C) 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D) 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E) 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT) assay. Alkaline phosphatase activity and osteocalcin (OC) dynamics were also measured.
BMP-4/7 alone significantly (P<0.05) promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent.
Interpretation & conclusions:
A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.
PMCID: PMC3767270  PMID: 24056563
Basic fibroblast growth factor; bone mesenchymal stromal cells; bone morphogenetic protein-4/7; bone tissue engineering
3.  Therapeutic Angiogenesis Using Basic Fibroblast Growth Factor in Combination with a Collagen Matrix in Chronic Hindlimb Ischemia 
The Scientific World Journal  2012;2012:652794.
Although therapeutic angiogenesis by angiogenic cytokines is a feasible strategy to improve regional blood flow in ischemic regions, the optimal delivery mode needs to be established. Here we designed a complex of collagen matrix (CM) and basic fibroblast growth factor (bFGF) and evaluated its proangiogenic effect in ischemic hindlimbs. The bFGF-CM was prepared using lyophilization. The morphology, porosity and toxicity of CM were examined. The bFGF releasing profile and bioactivity of released bFGF were assessed. bFGF-CM was intramuscularly implanted into the rabbit ischemic hindlimb model. Oxygen saturation parameters (OSP) of ischemic hindlimbs was measured to evaluate the extremity perfusion at intervals. Histological examination was performed to evaluate the level of angiogenesis. The CM and bFGF-CM were of identical multiporous structure lacking cytotoxicity. The releasing profile lasted 10 days and the released bFGF remained bioactive. OSP in bFGF-CM group was significantly higher than that in CM, bFGF and ischemic groups at 2 and 4 weeks. The number of capillaries and mature vessels in bFGF-CM group were significantly greater than that in untreated control, CM and bFGF groups. Therefore, bFGF-CM enables the safe and effective long-term release of bFGF with improved angiogenesis in ischemic hindlimbs compared with CM devoid of bFGF.
PMCID: PMC3362026  PMID: 22666143
4.  Effects of titania nanotubes with or without bovine serum albumin loaded on human gingival fibroblasts 
Modifying the surface of the transmucosal area is a key research area because this process positively affects the three functions of implants: attachment to soft tissue, inhibiting bacterial biofilm adhesion, and the preservation of the crestal bone. To exploit the potential of titania nanotube arrays (TNTs) with or without using bovine serum albumin (BSA) to modify the surface of a dental implant in contact with the transmucosal area, BSA was loaded into TNTs that were fabricated by anodizing Ti sheets; the physical characteristics of these arrays, including their morphology, chemical composition, surface roughness, contact angle, and surface free energy (SFE), were assessed. The effect of Ti surfaces with TNTs or TNTs-BSA on human gingival fibroblasts (HGFs) was determined by analyzing cell morphology, early adhesion, proliferation, type I collagen (COL-1) gene expression, and the extracellular secretion of COL-1. The results indicate that early HGF adhesion and spreading behavior is positively correlated with surface characteristics, including hydrophilicity, SFE, and surface roughness. Additionally, TNT surfaces not only promoted early HGF adhesion, but also promoted COL-1 secretion. BSA-loaded TNT surfaces promoted early HGF adhesion, while suppressing late proliferation and COL-1 secretion. Therefore, TNT-modified smooth surfaces are expected to be applicable for uses involving the transmucosal area. Further study is required to determine whether BSA-loaded TNT surfaces actually affect closed loop formation of connective tissue because BSA coating actions in vivo are very rapid.
PMCID: PMC3949701  PMID: 24623977
titania nanotubes; bovine serum albumin; modified surface; transmucosal area; human gingival fibroblast
5.  Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties 
Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization.
The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay.
CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells.
Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs. Quantitative analysis of angiogenesis at 14 days after implantation demonstrated that neovascularization of the implants containing SVF CD14+ cells or PB monocytes previously co-cultured with MSCs was 3.5 or 2 times higher than that observed in the implants with SVF-derived MSCs. Moreover, immunofluorescence of Matrigel sections revealed that SVF CD14+ cells differentiated into endothelial cells and contributed to vascular endothelium.
The results from this study suggest that adipose tissue-resident monocytes should contribute to tissue vascularization. Because SVF CD14+ cells were more efficient in inducing angiogenesis than SVF-derived MSCs, and differentiated into vascular endothelial cells, they may constitute a new cell source for cell-based therapeutic angiogenesis.
PMCID: PMC4055093  PMID: 24731246
6.  Increased fibroblast functionality on CNN2-loaded titania nanotubes 
Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor) fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.
PMCID: PMC3292419  PMID: 22403489
anodization; titania nanotubes; adhesion; connective tissue growth factor; fibroblast
7.  Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation 
The Journal of Cell Biology  1988;107(2):743-751.
Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.
PMCID: PMC2115214  PMID: 2971068
8.  Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican 
The Journal of Cell Biology  1996;133(2):405-416.
The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1) (Aviezer, D., D. Hecht, M. Safran, M. Eisinger, G. David, and A. Yayon. 1994. Cell 79:1005-1013). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan showed a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhanced the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that were not cotransfected with proteoglycan, but had no effect on this binding and decreased the yield of bFGFR cross-links in cells that were cotransfected with proteoglycan. Receptor-transfectant cells showed a decrease in glycophorin A expression when exposed to bFGF. This suppression was dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Finally, complementary cell- free binding assays indicated that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain was increased threefold when the syndecan- 4 ectodomain was coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, had no effect on this binding. We conclude that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness.
PMCID: PMC2120790  PMID: 8609172
9.  Anodized 20 nm diameter nanotubular titanium for improved bladder stent applications 
Materials currently used for bladder applications often suffer from incomplete coverage by urothelial cells (cells that line the interior of the bladder and ureter) which leads to the continuous exposure of the underlying materials aggravating an immune response. In particular, a ureteral (or sometimes called an ureteric or bladder) stent is a thin tube inserted into the ureter to prevent or treat obstruction of urine flow from the kidney. The main complications with ureteral stents are infection and blockage by encrustation, which can be avoided by promoting the formation of a monolayer of urothelial cells on the surface of the stent. Nanotechnology (or the use of nanomaterials) may aid in urothelialization of bladder stents since nanomaterials have been shown to have unique surface energetics to promote the adsorption of proteins important for urothelial cell adhesion and proliferation. Since many bladder stents are composed of titanium, this study investigated the attachment and spreading of human urothelial cells on different nanotextured titanium surfaces. An inexpensive and effective scaled up anodization process was used to create equally distributed nanotubular surfaces of different diameter sizes from 20–80 nm on titanium with lengths approximately 500 nm. Results showed that compared to untreated titanium stents and 80 nm diameter nanotubular titanium, 20 nm diameter nanotubular titanium stents enhanced human urothelial cell adhesion and growth up to 3 days in culture. In this manner, this study suggests that titanium anodized to possess nanotubular surface features should be further explored for bladder stent applications.
PMCID: PMC3075895  PMID: 21499419
anodization; nanotube; urothelial cells; bladder applications; titanium
10.  Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces 
Current orthopedic implants have functional lifetimes of only 10–15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone). To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin) and inflammation (dexamethasone) using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF). Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.
PMCID: PMC2527662  PMID: 18686785
anodization; titanium; adhesion; simulated body fluid; nanotubes; osteoblasts
11.  Self-Assembled Antimicrobial and biocompatible copolymer films on Titanium 
Macromolecular bioscience  2011;11(11):1515-1525.
Biofilm formation on biomedical devices such as dental implants can result in serious infections and finally in device failure. Polymer coatings which provide antimicrobial action to surfaces without compromising the compatibility with human tissue are of great interest. Copolymers of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate are interesting candidates in this respect. These copolymers form ultrathin polycationic layers on titanium surfaces. As the copolymerization reaction is almost ideal statistical, copolymers with varying compositions can be synthesized and immobilized onto titanium surfaces for comprehensive screening concerning antimicrobial activity and biocompatibility. Copolymer films on titanium were characterized by contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Antibacterial properties were assessed by investigation of adherence of S. mutans which represents a strain found in the human oral cavity. Biocompatibility was rated based on human gingival fibroblast adhesion, proliferation and cell morphology. Depending on polymer composition the coatings displayed a behavior ranging from biocompatibility equal to titanium but no antibacterial action to highly antimicrobial activity but poor biocompatibility. By balancing these two opposing effects by tailoring chemical composition, copolymer coatings were fabricated, which were able to inhibit the growth of S. mutans on the surface significantly but still show a sufficient attachment of gingival fibroblasts.
PMCID: PMC3784832  PMID: 21818855
antimicrobial polymer coatings; biocompatibility; copolymerization; medical implants; cell adhesion
12.  Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®) 
The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces.
Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR).
From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL.
Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels.
With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants.
PMCID: PMC4211057  PMID: 25352963
Enamel matrix derivative (Emdogain®); Human gingival fibroblast; Zirconia; Cell proliferation; Cell attachment
13.  In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles 
Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles.
Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco’s Modified Eagle’s Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites.
Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive.
PMCID: PMC3935549  PMID: 24578816
Adhesive; biocompatibility; cytotoxicity; nano-particles; orthodontics; titanium dioxide
14.  Inhibited bacterial biofilm formation and improved osteogenic activity on gentamicin-loaded titania nanotubes with various diameters 
Titania nanotubes loaded with antibiotics can deliver a high concentration of antibiotics locally at a specific site, thereby providing a promising strategy to prevent implant-associated infections. In this study we have fabricated titania nanotubes with various diameters (80, 120, 160, and 200 nm) and 200 nm length via electrochemical anodization. These nanotubes were loaded with 2 mg of gentamicin using a lyophilization method and vacuum drying. A standard strain, Staphylococcus epidermidis (American Type Culture Collection 35984), and two clinical isolates, S. aureus 376 and S. epidermidis 389, were selected to investigate the anti-infective ability of the gentamicin-loaded nanotubes (NT-G). Flat titanium (FlatTi) and nanotubes with no drug loading (NT) were also investigated and compared. We found that NT-G could significantly inhibit bacterial adhesion and biofilm formation compared to FlatTi or NT, and the NT-G with 160 nm and 200 nm diameters had stronger antibacterial activity because of the extended drug release time of NT-G with larger diameters. The NT also exhibited greater antibacterial ability than the FlatTi, while nanotubes with 80 nm or 120 nm diameters had better effects. Furthermore, human marrow derived mesenchymal stem cells were used to evaluate the effect of nanotubular topographies on the osteogenic differentiation of mesenchymal stem cells. Our results showed that NT-G and NT, especially those with 80 nm diameters, significantly promoted cell attachment, proliferation, spreading, and osteogenic differentiation when compared to FlatTi, and there was no significant difference between NT-G and NT with the same diameter. Therefore, nanotube modification and gentamicin loading can significantly improve the antibacterial ability and osteogenic activity of orthopedic implants.
PMCID: PMC3952900  PMID: 24634583
titania nanotubes; gentamicin; bacteria adhesion; biofilm formation; osteogenic activity
15.  Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures 
Molecular Biology of the Cell  1999;10(2):313-327.
Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF–2/GM1 interaction occurs with a Kd equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.
PMCID: PMC25171  PMID: 9950679
16.  Compositional analysis on heparin/heparan sulfate interacting with FGF•FGFR complexes 
Biochemistry  2009;48(35):8379-8386.
Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins thereby playing an essential role in the regulation of many physiological processes. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF•HS•FGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF or FGFR. In present report, we used a new strategy to study the structural specificity of HS binding to 10 different FGF•FGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desufated heparin and HS were used for the interaction studies by solution competition surface plasmon resonance (SPR) and using filter trapping assays. Specific oligosaccharides binding to FGF•FGFR complexes were subjected to polyacrylamide gel electrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography-mass spectrometry. The competition SPR studies using sized oligosaccharide mixtures showed that binding of each of the tested FGFs or FGF•FGFR complexes to heparin immobilized to an SPR chip were size dependent. The 6-desulfated heparin oligosaccharides showed reduced inhibition of FGF and FGF•FGFR binding to heparin in the competition experiments. Heparin and the 6-desulfated heparin showed higher inhibition to FGF•FGFR complex binding to heparin than to FGF binding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between the FGF•FGFR complexes and oligosaccharides. Disaccharide analysis showed HS disaccharides degree of polymerization (dp) 10 had high binding selectively, while heparin dp10 and 6-desulfated heparin dp10 showed reduced or no selectivity to the different FGF•FGFR complexes tested.
PMCID: PMC3348549  PMID: 19591432
17.  Investigation of biomaterials by human epithelial gingiva cells: an in vitro study 
Head & Face Medicine  2012;8:35.
In modern medicine and dentistry the use of biomaterials is a fast developing field of increasing interest. Especially in dentistry the interaction between biomaterials like implant materials and the soft tissue in the oral cavity is in the focus of daily research. In this context the high importance of testing materials and their surfaces concerning their biocompatibility towards corresponding cells is very likely. For this purpose this study investigates cells derived from human gingival biopsies on different materials and surfaces.
Cells in this study were cultivated out of human biopsies by a grow out explant technique and were sub cultivated on titanium, zirconium dioxide and collagen membrane specimens. To characterise the cells on the material surfaces used in this study immunohistochemical and histological staining techniques as well as different methods of microscopy (light microscopy and SEM) were applied.
With the aid of the explant technique and the chosen cell cultivation method it was possible to investigate the human gingiva derived cells on different materials. The data of the present study show that the human gingival cells attach and proliferate on all three tested materials by exhibiting characteristic gingival keratinocyte protein expression even after long periods of culture e.g. up to 70 days.
It could be shown that the three tested materials titanium, zirconium dioxide and collagen membrane (and their special surfaces) are good candidates for the application as materials in the dental gingival environment or, in the case of the collagen membrane as scaffold/cell-carrier for human gingival cells in tissue engineering.
PMCID: PMC3549823  PMID: 23241143
Human gingiva; Epithel; Biomaterials; Keratinocytes; In vitro study
18.  Tuning Cell Adhesion on Titanium with Osteogenic Rosette Nanotubes 
Self-assembled rosette nanotubes (RNTs), obtained from a twin G∧C base functionalized with lysine-arginine-serine-arginine (KRSR–(G∧C)2), were designed and investigated as bioactive coatings on titanium. These results were compared to RNTs derived from Lysine G∧C (K G∧C), Arg-Gly-Asp G∧C (RGD G∧C) and aminobutane–(G∧C)2 (AB–(G∧C)2). The results from this study revealed that these materials had excellent cytocompatibility properties as they enhanced osteoblast (bone forming cell) adhesion when coated on titanium. In particular, KRSR and RGD functionalized RNTs coated on titanium promoted the greatest osteoblast densities relative to untreated titanium. Furthermore, KRSR functionalized RNTs selectively improved osteoblast adhesion relative to fibroblast (soft-tissue forming cell) and endothelial cell adhesion. In contrast with these results, RNTs obtained from an unfunctionalized twin base (AB–(G∧C)2), RGD G∧C co-assembled with K G∧C and K-G∧C significantly enhanced endothelial cell attachment, which may find applications in the vascularization of newly formed bone tissue. In summary, these studies suggest that the surface of orthopedic implant materials (such as titanium) could be tailored to promote selective cell adhesion using biologically-inspired nanotubular structures functionalized with osteogenic compounds.
PMCID: PMC2943967  PMID: 20725961
rosette nanotubes; nanomaterials; biomimetic; osteogenic peptides; coating; orthopedic implant
19.  Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces 
BMC Oral Health  2014;14:75.
A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated.
The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium.
SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells.
Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to abutment surfaces in vivo.
PMCID: PMC4083866  PMID: 24952379
Oral keratinocytes; Gingival fibroblasts; Cell attachment; Dental implant; Surface modification; Oral bacteria
20.  Deciphering the mechanism behind Fibroblast Growth Factor (FGF) induced biphasic signal-response profiles 
The Fibroblast Growth Factor (FGF) pathway is driving various aspects of cellular responses in both normal and malignant cells. One interesting characteristic of this pathway is the biphasic nature of the cellular response to some FGF ligands like FGF2. Specifically, it has been shown that phenotypic behaviors controlled by FGF signaling, like migration and growth, reach maximal levels in response to intermediate concentrations, while high levels of FGF2 elicit weak responses. The mechanisms leading to the observed biphasic response remains unexplained.
A combination of experiments and computational modeling was used to understand the mechanism behind the observed biphasic signaling responses. FGF signaling involves a tertiary surface interaction that we captured with a computational model based on Ordinary Differential Equations (ODEs). It accounts for FGF2 binding to FGF receptors (FGFRs) and heparan sulfate glycosaminoglycans (HSGAGs), followed by receptor-phosphorylation, activation of the FRS2 adapter protein and the Ras-Raf signaling cascade. Quantitative protein assays were used to measure the dynamics of phosphorylated ERK (pERK) in response to a wide range of FGF2 ligand concentrations on a fine-grained time scale for the squamous cell lung cancer cell line H1703. We developed a novel approach combining Particle Swarm Optimization (PSO) and feature-based constraints in the objective function to calibrate the computational model to the experimental data. The model is validated using a series of extracellular and intracellular perturbation experiments. We demonstrate that in silico model predictions are in accordance with the observed in vitro results.
Using a combined approach of computational modeling and experiments we found that competition between binding of the ligand FGF2 to HSGAG and FGF receptor leads to the biphasic response. At low to intermediate concentrations of FGF2 there are sufficient free FGF receptors available for the FGF2-HSGAG complex to enable the formation of the trimeric signaling unit. At high ligand concentrations the ligand binding sites of the receptor become saturated and the trimeric signaling unit cannot be formed. This insight into the pathway is an important consideration for the pharmacological inhibition of this pathway.
PMCID: PMC4036111  PMID: 24885272
FGF signaling pathway; HSGAGs; Biphasic response; High throughput quantification; ODE-modeling; Particle swarm optimization
21.  Infection Mitigation Efficacy of Photoactive Titania on Orthopedic Implant Materials 
Advances in Orthopedics  2011;2011:571652.
In order to impede infection and achieve accelerated wound healing in the postorthopaedic surgery patients, a simple and benign procedure for creating nanotubular or nanofibrillar structure of photoactive TiO2 on the surface of Ti plates and wires is described. The nanoscale TiO2 films on titanium were grown by hydrothermal processing in one case and by anodization in the presence of dilute mineral acids under mild and benign conditions in the other. Confocal microscopy results demonstrated at least 50% reduction in the population of E. coli colonies (concentration 2.15 × 107 cells/mL) on TiO2-coated implants upon an IR exposure of up to 30 s; it required ∼20 min of exposure to UV beam for the same effect. These findings suggest the probability of eliminating wound infection during and after orthopedic surgical procedures by brief illumination of photoactive titania films on the implants with an IR beam.
PMCID: PMC3170812  PMID: 21994891
22.  αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells 
Molecular Biology of the Cell  1997;8(12):2449-2461.
Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.
PMCID: PMC25719  PMID: 9398667
23.  Experimental Polyvinyl Alcohol Core Coil for a Drug Delivery System 
Interventional Neuroradiology  2004;9(Suppl 1):107-111.
We developed a new type of coil with a polyvinyl alcohol core (PVA-core coil) to absorb and release various types of biologically active materials, for the endovascular treatment of intracranial aneurysms. A 10 mm segment of the PVA-core coil was used in this study. PVA-core coils were immersed in basic fibroblast growth factor (b-FGF) solution. The PVA-core coil, which absorbed b-FGF in the PVA core, was named FGF-core coil. This coil gradually released b-FGF in the solution without b-FGF. In vitro study, FGF-core coils, PVA-core coils and unmodified coils were cultured with fibroblasts (NIH3T3) respectively and their surface was observed with scanning electron microscopy (SEM). In vivo study, each coils were inserted into the rat common carotid artery. Rats were sacrificed and the arterial lumen were histologically examined 14 days and 28 days after coil implantation. Electron microscopy findings demonstrated remarkable cellular adhesion to the surface of the FGF-core coils, while no adhesion to the surface of the PVA-core coils and unmodified coils was found. Histologically, remarkable cellular proliferation and wall thickness like neointimal hyperplasia was demonstrated in the implanted common carotid artery of the FGF-core coil group at 14 days and 28 days. On the other hand, these changes did not occur in PVA-core coil group and unmodified coil group. We suggest that FGF-core coils may be effective to induce fibrotic changes inside cerebral aneurysms.
PMCID: PMC3553464  PMID: 20591238
polyvinyl alcohol, b-FGF, drug delivery, fibrosis
24.  Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening 
PLoS ONE  2012;7(3):e33999.
The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.
PMCID: PMC3307781  PMID: 22442738
25.  Blue-Violet Laser Modification of Titania Treated Titanium: Antibacterial and Osteo-Inductive Effects 
PLoS ONE  2013;8(12):e84327.
Many studies on surface modifications of titanium have been performed in an attempt to accelerate osseointegration. Recently, anatase titanium dioxide has been found to act as a photocatalyst that expresses antibiotic properties and exhibits hydrophilicity after ultraviolet exposure. A blue-violet semiconductor laser (BV-LD) has been developed as near-ultraviolet light. The purpose of this study was to investigate the effects of exposure to this BV-LD on surface modifications of titanium with the goal of enhancing osteoconductive and antibacterial properties.
The surfaces of pure commercial titanium were polished with #800 waterproof polishing papers and were treated with anatase titania solution. Specimens were exposed using BV-LD (λ = 405 nm) or an ultraviolet light-emitting diode (UV-LED, λ = 365 nm) at 6 mW/cm2 for 3 h. The surface modification was evaluated physically and biologically using the following parameters or tests: surface roughness, surface temperature during exposure, X-ray diffraction (XRD) analysis, contact angle, methylene blue degradation tests, adherence of Porphyromonas gingivalis, osteoblast and fibroblast proliferation, and histological examination after implantation in rats.
No significant changes were found in the surface roughness or XRD profiles after exposure. BV-LD exposure did not raise the surface temperature of titanium. The contact angle was significantly decreased, and methylene blue was significantly degraded. The number of attached P. gingivalis organisms was significantly reduced after BV-LD exposure compared to that in the no exposure group. New bone was observed around exposed specimens in the histological evaluation, and both the bone-to-specimen contact ratio and the new bone area increased significantly in exposed groups.
This study suggested that exposure of titanium to BV-LD can enhance the osteoconductivity of the titanium surface and induce antibacterial properties, similar to the properties observed following exposure to UV-LED.
PMCID: PMC3866166  PMID: 24358355

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