Epidermal growth factor-immobilized surfaces for the selective expansion of neural progenitor cells derived from induced pluripotent stem cells.

Neural progenitor cells (NPCs) are considered to be a promising source for stem cell-based regenerative therapy for central nervous disorders. However, the widespread clinical application of NPCs requires another technology that permits the efficient production of pure NPCs in large quantities. In this study, culture substrates were designed by immobilizing epidermal growth factor (EGF) onto the substrate and evaluated for their feasibility of expanding NPCs obtained through the neurosphere culture of induced pluripotent stem (iPS) cells. After three passages we obtained neurospheres that contained cells abundantly expressing an EGF receptor. The neurospheres were dissociated into single cells and seeded onto the EGF-immobilized substrates. It was observed that neurosphere-forming cells seeded and cultured on the EGF-immobilized surface formed a two-dimensional cellular network characteristic of NPCs. These cells were found to be capable of being subcultured, while remaining their proliferation potential. Furthermore, a majority of cells (~99% of total cells) on the substrate was shown to express an NPC marker, nestin, whereas a limited number of cells (~1% of total cells) expressed neuronal marker, ß-tubulin III. These results as a whole demonstrate that the EGF-immobilized substrate allows for iPS cell-derived NPCs to efficiently proliferate while maintaining the undifferentiated state.

Structure and function of engineered stromal cell-derived factor-1α.

To design biologically active, collagen-based scaffolds for bone tissue engineering, we have synthesized chimeric proteins consisting of stromal cell-derived factor-1α (SDF) and the von Willebrand factor A3 collagen-binding domain (CBD). The chimeric proteins were used to evaluate the effect of domain linkage and its order on the structure and function of the SDF and CBD. The structure of the chimeric proteins was analyzed by circular dichroism spectroscopy, while functional analysis was performed by a cell migration assay for the SDF domain and a collagen-binding assay for the CBD domain. Furthermore, computational structural prediction was conducted for the chimeric proteins to examine the consistency with the results of structural and functional analyses. Our structural and functional analyses as well as structural prediction revealed that linking two domains can affect their functions. However, their order had minor effects on the three-dimensional structure of CBD and SDF in the chimeric proteins.

Effect of laser engraving on shear bond strength of polyetheretherketone to indirect composite and denture-base resins.

Background/purpose: Polyetheretherketone (PEEK) is a highly sought-after thermoplastic due to its exceptional mechanical properties and biocompatibility. However, bonding PEEK to indirect composite resin (ICR) or denture-based resin (DBR) can be challenging. Laser engraving technology has shown potential to improve bonding for other materials; thus, this study aims to evaluate its effectiveness for PEEK. Materials and methods: The experiment involved preparing ingot-shaped PEEK samples, which were then categorized into four groups based on the treatment method employed: without treatment, air abrasion, sulfuric acid etching, and laser engraving (LS). Subsequently, the samples were bonded to ICR or DBR, and their shear bond strength (SBS) was tested with or without thermocycling using a universal testing machine. Furthermore, the failure mode was observed, with statistical analyses conducted to compare the results. Results: The grid-like microslit structure of LS group displayed the highest SBS for bonding PEEK to ICR or DBR (P < 0.05). During the bonding of PEEK to ICR, resin residue and penetration into the microslits were frequently observed in the LS group, indicating cohesive failure. However, when PEEK was bonded to DBR, mixture failure was frequently observed without thermocycling. After thermocycling, only the LS group showed cohesive failure, while the majority of specimens exhibited mixture failure. Conclusion: Laser engraving significantly improves the SBS between PEEK and both ICR and DBR. Furthermore, it was observed that resin had penetrated the microslits, indicating that laser engraving has great potential as a surface treatment method.

Nd:YVO4 laser groove treatment can improve the shear bond strength between dental PEEK and adhesive resin cement with an adhesive system.

The purpose of this study was to investigate the effect of various surface treatments on the shear bond strength between dental polyetheretherketone (PEEK) and adhesive resin cement. Two hundred and forty specimens were randomly classified into four groups: no treatment, sandblasted, sulfuric-acid-etched, and laser-grooved treatment. Each group was classified into two adhesive resin cement subgroups. Surface roughness, water contact angle, shear bond strength, and failure mode were measured; SEM and XPS results were obtained. The data were statistically analyzed using one-way or two-way analysis of variance and Tukey's honest significant difference test (α=0.05). Laser-grooved PEEK surface showed regular grooves and carbonization by thermal degradation; the surface roughness as well as water contact angle of were the highest in all groups. Shear bond strength values were significantly higher in the laser-groove-treated and sulfuric-acid-etched groups. Laser-groove-treated specimens showed cohesive failure. Laser-grooved treatment can improve shear bond strength between PEEK and adhesive resin cement.

Detection of synthetic RGDS(PO3H2)PA peptide adsorption using a titanium surface plasmon resonance biosensor.

The purpose of this study was to measure the time-dependent chemical interaction between synthetic RGDS(PO(3)H(2))PA (P-RGD) peptide and titanium surfaces using a titanium surface plasmon resonance (SPR) biosensor and to determine the degree of peptide immobilization on the surfaces. An SPR instrument for 'single-spot' analysis was used for nanometer-scale detection of biomolecular adsorption using a He-Ne laser light according to Knoll's method. The oxidized titanium surface was etched when exposed to H(3)PO(4) solutions with a pH of 2.0 or below. The amount of P-RGD adsorbed at pH 1.9 was approximately 3.6 times as much as that at pH 3.0 (P < 0.05). P-RGD naturally adsorbed on the oxidized titanium surface as a consequence of the bonding and dissociation mechanism of the phosphate functional group. Furthermore, the control of pH played a very important role in the interaction between P-RGD and the surface. These findings show that pH control may promote progressive binding of biomolecules with the phosphate functional group to the titanium surface.

Evaluation of a peptide motif designed for protein tethering to polymer surfaces.

In search for peptide motifs that allow us to efficiently tether fusion proteins onto polymer surfaces, we designed a KLKLKLKLKL (KL5) decapeptide in which basic and hydrophobic amino acids were alternately linked. By means of genetic engineering technology together with a bacterial expression system, the KL5 fusions of epidermal growth factor (EGF), basic fibroblast growth factor, and stromal cell-derived factor-1α were prepared together with their control counterparts without KL5. The adsorption experiments were performed for these fusion proteins on the surface of polystyrene, hydrophilized polystyrene, and polycaprolactone by surface plasmon resonance analysis. To understand the results of the binding assays, the structure of the fusion proteins was predicted by ab initio computer simulation and analyzed empirically by circular dichroism spectroscopy. The result of structural analyses suggested that the KL5 peptide is exposed to the outside and has a negligible effect on the structure of the protein partners. However, it was found that the efficiency of KL5 as a peptide motif greatly depends on protein partners. Our results showed that KL5 exerts most effectively its function as a peptide motif when fused to acidic proteins such as EGF. Indeed, the number of living human mesenchymal stem cells determined after 7-day culture was larger on the polystyrene and polycaprolactone surfaces with EGF tethered through the KL5 peptide than control surfaces. According to the results obtained in this study, we conclude that KL5 is useful as a peptide motif for tethering a specific class of protein partners.

Oriented immobilization of basic fibroblast growth factor: Bioengineered surface design for the expansion of human mesenchymal stromal cells.

E. coli expressed recombinant basic fibroblast growth factor (bFGF) with histidine-tag (bFGF-His) was immobilized onto the surface of a glass plate modified with a Ni(II)-chelated alkanethiol monolayer. The immobilization is expected to take place through the coordination between Ni(II) and His-tag. The bFGF-immobilized surface was exposed to citrate buffer solution to refold in situ the surface-immobilized bFGF. The secondary structure of immobilized bFGF-His was analyzed by solid-phase circular dichroism (CD) spectroscopy. Immortalized human mesenchymal stromal cells (hMSCs) were cultured on the bFGF-His-immobilized surface to examine their proliferation. CD spectroscopy revealed that the immobilized bFGF initially exhibited secondary structure rich in α-helix and that the spectrum was gradually transformed to exhibit the formation of ß-strands upon exposure to citrate buffer solution, approaching to the spectrum of native bFGF. The rate of hMSC proliferation was 1.2-fold higher on the bFGF-immobilized surface treated with in situ citrate buffer, compared to the polystyrene surface. The immobilized bFGF-His treated in situ with citrate buffer solution seemed to be biologically active because its secondary structure approached its native state. This was well demonstrated by the cell culture experiments. From these results we conclude that immobilization of bFGF on the culture substrate serves to enhance proliferation of hMSCs.

Bone formation ability of carbonate apatite-collagen scaffolds with different carbonate contents.

Hydroxyapatite and carbonate apatites with different carbonate contents were synthesized, mixed with atelocollagen, and made into sponge scaffolds. The scaffolds were implanted into the bone sockets of the femurs of male New Zealand white rabbits for 2, 3, 12 and 24 weeks. carbonate apatite-collagen scaffold with 4.8 wt% carbonate content appeared to have similar crystallinity and chemical composition to human bone. When the scaffolds were implanted into the rabbit femurs, histological observation indicated that the carbonate apatites-collagen scaffolds with relatively higher carbonate contents were gradually deformed throughout the implantation period, and showed uniform surrounding bone after 24 weeks and could not be distinguished. The carbonate apatite-collagen scaffold with 4.8 wt% carbonate content showed the highest bone area ratio of all of the scaffolds. It is suggested that a carbonate apatite-collagen scaffold with carbonate content similar to that of human bone may have optimal bone formation ability.

Effect of microslit retention on the bond strength of zirconia to dental materials.

The aim of this study was to investigate the effect of microslits formed by Nd:YVO4 laser beam machining on the bond strength between two types of zirconia, yttria-partially stabilized zirconia (Y-TZP) and ceria-partially stabilized zirconia/alumina nanocomposite (Ce-TZP/A), and porcelain or two types of resin. Zirconia disks were divided into three groups: 1) non-treated (NT); 2) blasted with alumina particles (AB); 3) microslits fabricated on a zirconia surface by laser beam machining (MS). After veneering porcelain or resins on zirconia specimens, halves of the resin specimens were thermocycled up to 20,000 cycles. The shear bond strength between porcelain and both types of zirconia was not improved by the microslits. Before and after thermocycling, the bond strength between an indirect composite resin or acrylic resin and Y-TZP with microslits was the highest. It was concluded that the microslits on Y-TZP enabled micromechanical interlocking and improved the bond strength and durability of the resins.

Effect of laser groove treatment on shear bond strength of resin-based luting agent to polyetheretherketone (PEEK).

PURPOSE: The mechanical properties of polyetheretherketone (PEEK) are ideally suited for fixed dental prostheses. However, PEEK typically has low adhesion strength to resin-based luting agent. This study assessed the shear bond strength between laser groove treated PEEK and resin-based luting agent. METHODS: A total of 230 specimens were randomly divided into five groups (n=46): no-treatment, air abrasion treatment, 100µm-deep, 150µm-deep, and 200µm-deep laser groove treatments. The surface roughness was measured, scanning electron microscopy was used to observe the specimen surfaces, and X-ray photoelectron spectroscopy (XPS) was used to analyze the surfaces. Each group was divided into four resin-based luting agent subgroups: Panavia V5, RelyX Ultimate Resin Cement, G-CEM Link Force, and Super-Bond C&B. After the resin-based luting agent was bonded to the specimens, the bond strength was measured using shear tests and the failure modes were assessed by stereomicroscopy. The surfaces were also observed by scanning electron microscopy (SEM) after the shear bond strength measurements. The data were statistically analyzed using a two-way analysis of variance and Tukey's honest significant difference test (α=0.05). RESULTS: The PEEK surface after laser groove treatment groups exhibited the highest mean Ra values. In the XPS analysis, the laser treated PEEK surface exhibited an effective surface composition for bonding with resin-based luting agent. The shear bond strengths for the laser groove treated samples were significantly higher (p<0.05) than those of the no-treatment and air abrasion treatment groups. CONCLUSIONS: The shear bond strength between PEEK and resin-based luting agent was substantially improved by laser groove treatment.

Complement activation on surfaces carrying amino groups.

The complement system is strongly activated by surfaces carrying nucleophilic groups, such as hydroxyl (OH) groups, and triggered by deposition of complement protein fragment, C3b. Surfaces carrying amino groups, the other representative nucleophilic group, are expected to be potential activators of the complement system through the alternative pathway. Few studies thus far have examined the potential of artificial materials carrying amino groups in activating the complement system. In this study, we employed a self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (NH2-SAM) and a polyethyleneimine (PEI)-coated surface as model surfaces to study interactions between amino groups and serum complement pathway. SAMs of 11-mercaptoundecanol (OH-SAM) and 1-dodecanethiol (CH3-SAM) were used as control surfaces, respectively. Although much protein was adsorbed from serum solutions on the two types of amino surfaces, amounts of C3b deposition were much less than those observed on OH-SAM. Amounts of C3a released on the amino surfaces were same levels as that of CH3-SAM, but significantly smaller than that on OH-SAM. These facts suggest that the nucleophilic amino groups on NH2-SAM and PEI-coated surfaces do not directly activate the alternative pathway, but the protein adsorbed layers formed on amino surfaces activate it, but to an extent much smaller than that on OH-SAM. In addition, we found no deposition of C1q molecules on the amino surfaces, suggesting that these surfaces fail to activate the classical pathway. However, more careful studies are needed to conclude it, because it is known that C1q is only transiently detected at typical classical activation interfaces.

Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts.

OBJECTIVES: The effects of chlorine dioxide (ClO2), sodium hypochlorite (NaOCl), and hydrogen peroxide (H2O2) on cell death and the cell cycle of human gingival fibroblast (HGF) cells were examined. METHODS: The inhibition of HGF cell growth was evaluated using a Cell Counting Kit-8. The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G0/G1, S, and G2/M phases) using flow cytometry. The patterns of cell death (necrosis and apoptosis) were analyzed using flow cytometry with annexin V-FITC/PI staining. RESULTS: The lethal doses for 50% of the cells (LD50) of ClO2, NaOCl, and H2O2 were 0.16, 0.79, and 0.11 mM, respectively. All three dental disinfectants induced G0/G1 cell cycle arrest. H2O2 induced apoptosis at concentrations of 0.05 and 0.1 mM, while NaOCl and ClO2 did not induce significant apoptosis at any concentration examined. CONCLUSIONS: These results suggest that ClO2 is sufficient for use as a dental disinfectant compared with H2O2 or NaOCl.

Formation of chemical bonds on zirconia surfaces with acidic functional monomers.

We investigated the chemical interaction between zirconia surfaces and functional monomers using X-ray photoelectron spectroscopy (XPS). Two types of zirconia disks cleaned with piranha solution were treated with one of two phosphate primers (Alloy Primer, Clearfil Ceramic Primer) or a carboxylic primer (Super-Bond C&B Monomer), and rinsed 3 times with acetone. XPS analysis revealed that phosphorus was incorporated into zirconia when the surface was treated with a primer containing phosphate monomer (10-methacryloyloxydecyl dihydrogen phosphate; MDP). However, the S 2p peak of a triazine dithiol monomer (6-[N-(4-vinylbenzyl)-n-propylamino]-1,3,5-triazine-2,4-dithione; VTD) and Si 2p peak of silane (3-trimethoxysilylpropyl methacrylate; TMSPMA) were not detected in the spectra of the primed surface. The [C]/[Zr] ratio for the surface treated with a carboxylic anhydride (4-methacryloyloxyethyl trimellitate anhydride; 4-META) primer was smaller than that treated with MDP. These results demonstrated that 4-META and MDP adsorbed to zirconia, whereas the VTD and TMSPMA did not.

Enhancement of calcification by osteoblasts cultured on hydroxyapatite surfaces with adsorbed inorganic polyphosphate.

Inorganic polyphosphate has been expected to accelerate bone regeneration. However, there are limited evidences to prove that polyphosphate adsorbed on the surface of a hydroxyapatite plate enhances calcification of cultured osteoblasts. In this study, we examined the effect of polyphosphate adsorbed onto the surface of a hydroxyapatite plate on the attachment, proliferation, differentiation, and calcification of osteoblasts. After hydroxyapatite plates were soaked in solutions of polyphosphate, the plate surfaces were analyzed by scanning electron microscopy and toluidine blue staining to confirm adsorption of polyphosphate. The hydroxyapatite plates were further subjected to the measurements of surface roughness, water contact angle, and the binding capacity of calcium ions. Cell culture experiments were carried out using MC3T3-E1 pre-osteoblastic cells. It was found that soaking a hydroxyapatite plate in a polyphosphate solution gave rise to an increase in surface roughness and reduction in water contact angle in a concentration-dependent manner, suggesting the adsorption of polyphosphate onto the surface of a hydroxyapatite plate. It was further observed that surface-adsorbed polyphosphate exhibited an inhibitory effect on cell adhesion and proliferation. In contrast, cell differentiation was promoted on hydroxyapatite plates with adsorbed polyphosphate, when assessed from expression of differentiation marker genes including alkaline phosphatase, osteopontin, and osteocalcin. In addition, calcification of the culture was enhanced on hydroxyapatite plates with relatively low density of adsorbed polyphosphate. Our results as a whole provided an evidence to show that there is a narrow window with regard to the surface density of adsorbed polyphosphate for the enhancement of osteoblast calcification.

Effect of ozonated water on the surface roughness of dental stone casts.

Infection control of dental stone cast is an important issue. Ozone is effective for disinfection against microorganisms and inactivation of viruses. However, there is little information regarding the use of ozone. We prepared 4 types of gypsum specimens and 3 types of disinfectants (4-5 ppm Ozonated water [OZW], 2% glutaraldehyde [GL], and 1% sodium hypochlorite [SH]). Gypsum specimens were immersed in each disinfectant for 5 and 10 min, and surface roughness was then examined using laser scanning microscopy. Surface microstructure was investigated using scanning electron microscopy. Immersion of gypsum specimens in SH, GL, and OZW increased the surface roughness to a maximum of 1.04, 0.37, and 0.30 µm, respectively, based on the difference between the average values of surface roughness before and after the disinfection procedure. The effects of OZW and GL were comparable. OZW is useful as a candidate for relatively safe disinfection of material for dental stone casts.

Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation.

Neural stem cell (NSC) has emerged as a potential source for cell replacement therapy following traumatic injuries and degenerative diseases of the central nervous system. However, clinical applications of NSC further require technological advances especially for controlling differentiation of NSC. This study aimed at developing biomaterials that serve to expand undifferentiated NSC or to induce cells with specific phenotypes. Our approach is to construct composite biomaterials that consist of extracellular matrix components and growth factors. In order to optimize matrix-growth factor combinations, we conducted the parallel and rapid screening of composite biomaterials through assays using cell-based arrays. The photo-assisted patterning of an alkanethiol self-assembled monolayer was employed to achieve site-addressable combinatorial immobilization of natural and synthetic matrices incorporated with growth factors including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). NSC obtained from the rat embryonic striatum was cultured directly on the array to screen for cell adhesion, proliferation, and promotion of neuronal and glial specification. The results showed that the significant number of cells adhered to laminin-1, fibronectin, ProNectin, and poly(ethyleneimine). It was found that cells proliferated most extensively on a spot with immobilized EGF among the spots with different matrix-growth factor combinations. The results also showed that neuronal differentiation was promoted on the spots with immobilized NGF or NT-3, and astroglial differentiation with CNTF. Importantly, observed effects of growth factors were frequently altered depending on the type of co-immobilized matrices, suggesting synergic effects of adhesion and growth factor signals.

Cell adhesion and proliferation patterns on mixed self-assembled monolayers carrying various ratios of hydroxyl and methyl groups.

Mixed self-assembled monolayers (SAMs) with hydroxyl and methyl surface groups were prepared as biomaterial surface models which were well-controlled and well-defined. The surface properties of mixed SAMs were examined by X-ray photoelectron spectroscopy and water contact angle measurement. It was found that the parameter of water contact angle more accurately reflected the surface compositions of mixed SAMs than by the mixing ratio of the two alkanethiols. Cell adhesion and growth were also examined on mixed SAMs of various wettability conditions. It was found that amount of serum protein adsorption changed with the surface composition. To examine the effect of surface composition on cell growth pattern, four cell types--C3H10T1/2-clone 8, L929, UVB6-2.1A, and MC3T3E1--were incubated on mixed SAMs for three or six days. Differences in cell growth pattern against wettability were clearly recognized for each cell type. In light of the results obtained in this study, the relationship between the biocompatibility of biomaterials and surface factors were thus clarified.

Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures.

The aim of this study was to examine the apoptotic and necrotic influence of four dental resin polymerization initiators--namely benzoyl peroxide (BPO), camphorquinone (CQ), dimethylaminoethyl methacrylate (DMAEMA), and dimethyl-para-toluidine (DMPT)--on human gingival fibroblast (HGF) cells. To this end, the growth inhibition of HGF cells with 1 mM BPO, CQ, and DMAEMA, and 500 microM DMPT was evaluated using Cell Counting Kit-8. Then, cell cycle analysis by flow cytometry was used to assess propidium iodide-stained cells (distribution of cells in G0/G1, S, G2/M phases). All four dental resin polymerization initiators induced G0/G1 cell cycle arrest. As for the patterns of cell death (necrosis and/or apoptosis), they were analyzed using Annexin V-FITC/PI staining with flow cytometry. All four dental resin polymerization initiators most likely induced necrosis.

Immobilization of simulated reducing agent at the surface of SiO2 fillers in dental composite resins.

To reduce the leachability of reducing agents from composite resins, immobilization of a simulated reducing agent at the surface of SiO2 fillers was examined. SiO2 plates were immersed in 2% 3-aminopropyltriethoxy silane/ethanol solution, and then immersed in dimethyl sulfoxide with 0.25 wt% 4-dimethyl amino benzoic acid (DMABA), 2.0 wt% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 0.5 wt% N-hydroxysuccinimide. Wide-scan spectrum of X-ray photoelectron spectroscopy did not detect carbon contamination. However, narrow scan detected an O=C-N peak at 399.8 eV, suggesting that DMABA could be immobilized on silane-coupled SiO2 plates. Further, surface plasmon resonance analysis indicated the adsorption of MMA at the surface of reducing agent-immobilized plate.

Synthetic osteopontin-derived peptide SVVYGLR can induce neovascularization in artificial bone marrow scaffold biomaterials.

We have previously reported that an osteopontin-derived SVVYGLR peptide exhibited potent angiogenic activity in vitro and in vivo. In the present study, the focus points were on the in vitro effect of SVVYGLR on bone marrow stromal cell proliferation, as well as its in vivo effect on bone tissue formation when grafts made of CO3Ap-collagen sponge- as a scaffold biomaterial containing the SVVYGLR motif - were implanted. SVVYGLR peptide promoted bone marrow stromal cell proliferation. When a CO3Ap-collagen sponge containing SVVYGLR peptide was implanted as a graft into a tissue defect created in rat tibia, the migration of numerous vascular endothelial cells - as well as prominent angiogenesis - inside the graft could be detected after one week. These results thus suggested that our scaffold biomaterials including the peptide could be useful for bone tissue regeneration.