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.

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.

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.

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.

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.

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.

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.

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.

The synthesis and characterization of a novel potassium chloride-fluoridated hydroxyapatite varnish for treating dentin hypersensitivity.

Dentin hypersensitivity is treated using materials that occlude the dentinal tubules or release potassium ions that induce nerve desensitization. In this study we formulated a novel varnish containing potassium chloride and fluoridated hydroxyapatite and evaluated its physical properties and cytotoxicity. Potassium ion release from the varnish was measured. Dentin permeability was evaluated by measuring the hydraulic conductance of etched dentin discs treated with the varnish. The direct contact test and MTT assay were performed to evaluate the varnish's cytotoxicity. We found that the varnish released potassium ions over 6 h, and demonstrated a statistically higher reduction in dentin permeability compared to commercial fluoride varnish or control. Dentin disc scanning electron microscopy images demonstrated occluded dentinal tubules in the novel varnish group after brushing. The cytotoxicity tests indicated the varnish was biocompatible with gingival and pulpal fibroblasts. We propose the novel varnish is a potential material for use in hypersensitivity management.

Effect of thione primers on adhesive bonding between an indirect composite material and Ag-Pd-Cu-Au alloy.

The current study evaluated the effect of primers on the shear bond strength of an indirect composite material joined to a silverpalladium-copper-gold (Ag-Pd-Cu-Au) alloy (Castwell). Disk specimens were cast from the alloy and were air-abraded with alumina. Eight metal primers were applied to the alloy surface. A light-polymerized indirect composite material (Solidex) was bonded to the alloy. Shear bond strength was determined both before and after the application of thermocycling. Two groups primed with Metaltite (thione) and M. L. Primer (sulfide) showed the greatest post-thermocycling bond strength (8.8 and 6.5 MPa). The results of the X-ray photoelectron spectroscopic (XPS) analysis suggested that the thione monomer (MTU-6) in the Metaltite primer was strongly adsorbed onto the Ag-Pd-Cu-Au alloy surface even after repeated cleaning with acetone. The application of either the thione (MTU-6) or sulfide primer is effective for enhancing the bonding between a composite material and Ag-Pd-Cu-Au alloy.

Bioactive surface modification of hydroxyapatite.

The purpose of this study was to establish an acid-etching procedure for altering the Ca/P ratio of the nanostructured surface of hydroxyapatite (HAP) by using surface chemical and morphological analyses (XPS, XRD, SEM, surface roughness, and wettability) and to evaluate the in vitro response of osteoblast-like cells (MC3T3-E1 cells) to the modified surfaces. This study utilized HAP and HAP treated with 10%, 20%, 30%, 40%, 50%, or 60% phosphoric acid solution for 10 minutes at 25°C, followed by rinsing 3 times with ultrapure water. The 30% phosphoric acid etching process that provided a Ca/P ratio of 1.50, without destruction of the grain boundary of HAP, was selected as a surface-modification procedure. Additionally, HAP treated by the 30% phosphoric acid etching process was stored under dry conditions at 25°C for 12 hours, and the Ca/P ratio approximated to 1.00 accidentally. The initial adhesion, proliferation, and differentiation (alkaline phosphatase (ALP) activity and relative mRNA level for ALP) of MC3T3-E1 cells on the modified surfaces were significantly promoted (P < 0.05 and 0.01). These findings show that the 30% phosphoric acid etching process for the nanostructured HAP surface can alter the Ca/P ratio effectively and may accelerate the initial adhesion, proliferation, and differentiation of MC3T3-E1 cells.

New development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering.

Carbonate apatite-chitosan scaffolds (CA-ChSs) were fabricated using the lyophilization technique. It was found that ChSs prepared with 200 mg chitosan powder (ChSs200) had well-structured three-dimensional architecture with high porosity and good retentive form without brittleness. In addition, it was shown that the number of osteoblast-like cells MC3T3-E1 proliferated on desalinated ChSs200 was larger than that on the non-desalinated ChSs200. CA-ChSs were fabricated by adding 100 mg carbonate apatite (CA) to 200 mg chitosan gels followed by freeze-drying (CA100ChSs200). SEM observation revealed that CA100ChSs200 had favorable three- dimensional porous structures. The number of living cells increased more rapidly on CA100ChSs200 prepared with different amounts of CA than on ChSs. ALP activity significantly increased after day 14 and reached a plateau after day 21 in ChSs200 and CA100ChSs200. It was concluded that newly developed CA100ChSs200 may be a possible scaffold material for tissue engineering.

Influence of alkyl chain length on calcium phosphate deposition onto titanium surfaces modified with alkylphosphonic acid monolayers.

Much attention has been paid to the modification of a titanium surface with an alkylphosphonic acid (PA)-based self-assembled monolayer (SAM) to accelerate hydroxyapatite (HA) deposition on the surface. In order to further accelerate the rate of HA deposition, we examined here the effect of alkyl chain length of SAMs on the formation of a HA layer. PAs with three different alkyl chain lengths (3, 6, and 16 methylene units) were used for the preparation of a SAM on titanium. The titanium specimens with monolayers were soaked in a simulated body fluid under physiological conditions for 4 weeks. The deposited substances were analyzed by scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. These analyses revealed that the formation of PA SAMs accelerate the deposition of poorly crystallized HA, in an alkyl chain length-dependent manner. Among PAs studied here, PA containing a 16-carbon alkyl chain gave rise to the titanium surface most effective for the deposition of HA.

A novel glass ionomer cement containing MgCO(3 )apatite induced the increased proliferation and differentiation of human pulp cells in vitro.

This study aimed to investigate the in vitro biological response of human dental pulp cells to glass ionomer cement (GIC, Fuji IX GP(®)) containing 2.5% magnesium carbonate apatite (MgCO(3)Ap). MgCO(3)Ap was synthesized by wet method and characterized using FT-IR, XPS, and SEM. Fuji IX GP(®) served as a control. Test and control cements were prepared by encapsulated mixing the powder with Fuji IX-liquid (P/L=3.6:1). Eluates from cements extracted by 1 mL culture medium were collected at day 1, 7 and 14, and used for WST-1 proliferation assay. For ALPase activity, cells were maintained with cements in transwells, harvested and enzyme activity was measured at day 1, 4, 7, 14, and 21. We found a higher cell proliferation and increased ALPase activity by pulp cells in the test group compared to the control. This suggests the potential of GIC containing this novel biological apatite as a restorative material for pulp-dentin regeneration.

Real time assessment of surface interactions with a titanium passivation layer by surface plasmon resonance.

Due to the high corrosion resistance and strength to density ratio titanium is widely used in industry, and also in a gamut of medical applications. Here we report for the first time on our development of a titanium passivation layer sensor that makes use of surface plasmon resonance (SPR). The deposited titanium metal layer on the sensor was passivated in air, similarly to titanium medical devices. Our "Ti-SPR sensor" enables analysis of biomolecule interactions with the passivated surface of titanium in real time. As a proof of concept, corrosion of a titanium passivation layer exposed to acid was monitored in real time. The Ti-SPR sensor can also accurately measure the time-dependence of protein adsorption onto the titanium passivation layer at sub-nanogram per square millimeter accuracy. Besides such SPR analyses, SPR imaging (SPRI) enables real time assessment of chemical surface processes that occur simultaneously at "multiple independent spots" on the Ti-SPR sensor, such as acid corrosion or adhesion of cells. Our Ti-SPR sensor will therefore be very useful to study titanium corrosion phenomena and biomolecular titanium-surface interactions with application in a broad range of industrial and biomedical fields.

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.

Degree of immobilization of synthetic RGDS(PO(3)H(2))PA peptides on titanium surfaces.

The purpose of this study was to characterize the chemical interaction between titanium surfaces and the peptide RGDS(PO(3)H(2))PA (P-RGD) synthesized from RGD peptide (RGD) and o-phospho-L-serine (P-Ser), and to determine the degree of peptide immobilization on the titanium surface. X-ray photoelectron spectroscopy showed that the adsorption amount of RGD was significantly smaller than those of P-Ser and P-RGD (p<0.05). Furthermore, although it appeared that P-RGD bonded to the surface, ultrasonic rinsing with water caused it to dissociate, releasing RGD and leaving only S(PO(3)H(2))PA bonded to the surface. These findings show that although it remains difficult to obtain a stable P-RGD layer, the phosphate functional group greatly improves immobilization of the molecule on titanium surfaces.