Mariko Nakamura

Nano-controlled molecular interaction at adhesive interfaces for hard tissue reconstruction

Although decayed/fractured teeth can be reconstructed minimally invasively and nearly invisibly using adhesive technology, the clinical longevity of dental composite restorations is still too short. Water sorption is thought to be the principal cause of destabilization of the biomaterial-tooth bond. However, the actual mechanisms of interfacial degradation are far from understood. Here we report how nano-controlled molecular interaction at the biomaterial-hard tissue interface can improve bond durability. The use of functional monomers with a strong chemical affinity for the calcium in hydroxyapatite is essential for long-term durability. Correlative X-ray diffraction and solid-state nuclear magnetic resonance disclosed a time-dependent molecular interaction at the interface with stable ionic bond formation of the monomer to hydroxyapatite competing in time with the deposition of less stable calcium phosphate salts. The advanced tooth-biomaterial interaction model gives not only an insight into the mechanisms of bond degradation, but also provides a basis to develop functional monomers for more durable tooth reconstruction.

Development of a,b-plane-oriented hydroxyapatite ceramics as models for living bones and their cell adhesion behavior

In vertebrate bones and tooth enamel surfaces, the respective a,b-planes and c-planes of hydroxyapatite (HAp) crystals are preferentially exposed. However, the reason why the HAp crystals show different orientations depending on the type of hard tissues is not yet understood. To clarify this question, appropriate ceramic models with highly preferred orientation are necessary. In the present study, dense HAp ceramic models which have the same orientation as living bones were fabricated using composite powders of c-axis-oriented single-crystal apatite fibers (AF) and wet-synthesized apatite gels (AG). The results of crystalline identification and ultrastructural observation showed that the resulting HAp ceramics maintained the c-axis orientation of the AF particles, and their high a,b-plane orientation degrees could be maintained with small additive amounts of AG; however, when the AG content was over 30 mass%, this value decreased. The influence of orientation degree on the surface characteristics was investigated by evaluating the surface zeta-potential and wettability. These results show that increasing the a,b-plane orientation degree shifted the surface charge from negative to positive, and decreased the surface wettability. Initial cell-attachment assays were performed on these resulting ceramics using MC3T3-E1 cells as models of osteoblasts. The results show that the cell-attachment efficiency decreased with increasing a,b-plane orientation degree.

A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering

Osteochondral injuries remain difficult to repair. We developed a novel photo-cross-linkable furfurylamine-conjugated gelatin (gelatin-FA). Gelatin-FA was rapidly cross-linked by visible light with Rose Bengal, a light sensitizer, and was kept gelled for 3 weeks submerged in saline at 37°C. When bone marrow-derived stromal cells (BMSCs) were suspended in gelatin-FA with 0.05% Rose Bengal, approximately 87% of the cells were viable in the hydrogel at 24 h after photo-cross-linking, and the chondrogenic differentiation of BMSCs was maintained for up to 3 weeks. BMP4 fusion protein with a collagen binding domain (CBD) was retained in the hydrogels at higher levels than unmodified BMP4. Gelatin-FA was subsequently employed as a scaffold for BMSCs and CBD-BMP4 in a rabbit osteochondral defect model. In both cases, the defect was repaired with articular cartilage-like tissue and regenerated subchondral bone. This novel, photo-cross-linkable gelatin appears to be a promising scaffold for the treatment of osteochondral injury.

Visible light-induced crosslinkable gelatin

A novel visible light-crosslinkable porcine gelatin was prepared for gelation and micropatterning. The preparation employed a photo-oxidation-induced crosslinking mechanism. First, furfuryl groups were incorporated into the gelatin. Second, the modified gelatin was mixed in water with Rose Bengal, which is a visible light sensitizer. Irradiation by visible light solidified the aqueous solution. In addition, when the solution was cast on a plate, dried and photo-irradiated in the presence of a photomask a micropattern was formed that matched the micropattern on the photomask. The gelatin-immobilized regions enhanced cell adhesion. It was also confirmed that the gelatin incorporating furfuryl and Rose Bengal have no significant toxicity. The photo-crosslinkable gelatin was employed as a direct pulp capping material in the dental field. Considering these results, this system could be useful as a new type of visible light-induced crosslinkable biosealant.

Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4

Purpose Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein. Methods BMP4 was fused with a nanosized carrier, collagen-binding domain (CBD), derived from fibronectin. The stability of the CBD-BMP4 fusion protein was examined in vitro and in vivo. Osteogenic effects of CBD-BMP4 were evaluated by computer tomography after intramedullary injection without a collagen–sponge scaffold. Recombinant BMP-4, CBD, or vehicle were used as controls. Expressions of bone-related genes and growth factors were compared among the groups. Osteogenesis induced by CBD-BMP4, BMP4, and CBD was also assessed in a bone-defect model. Results In vitro, CBD-BMP4 was retained in a collagen gel for at least 7 days while BMP4 alone was released within 3 hours. In vivo, CBD-BMP4 remained at the given site for at least 2 weeks, both with or without a collagen–sponge scaffold, while BMP4 disappeared from the site within 3 days after injection. CBD-BMP4 induced better bone formation than BMP4 did alone, CBD alone, and vehicle after the intramedullary injection into the mouse femur. Bone-related genes and growth factors were expressed at higher levels in CBD-BMP4-treated mice than in all other groups, including BMP4-treated mice. Finally, CBD-BMP4 potentiated more bone formation than did controls, including BMP4 alone, when applied to cranial bone defects without a collagen scaffold. Conclusion Altogether, nanocarrier-CBD enhanced the retention of BMP4 in the bone, thereby promoting augmented osteogenic responses in the absence of a scaffold. These results suggest that CBD-BMP4 may be clinically useful in facilitating bone formation.

Novel Fluoro-carbon Functional Monomer for Dental Bonding:

Among several functional monomers, 10-methacryloxydecyl dihydrogen phosphate (10-MDP) bonded most effectively to hydroxyapatite (HAp). However, more hydrolysis-resistant functional monomers are needed to improve bond durability. Here, we investigated the adhesive potential of the novel fluoro-carbon functional monomer 6-methacryloxy-2,2,3,3,4,4,5,5-octafluorohexyl dihydrogen phosphate (MF8P; Kuraray Noritake Dental Inc., Tokyo, Japan) by studying its molecular interaction with powder HAp using solid-state nuclear magnetic resonance (1H MAS NMR) and with dentin using x-ray diffraction (XRD) and by characterizing its interface ultrastructure at dentin using transmission electron microscopy (TEM). We further determined the dissolution rate of the MF8P_Ca salt, the hydrophobicity of MF8P, and the bond strength of an experimental MF8P-based adhesive to dentin. NMR confirmed chemical adsorption of MF8P onto HAp. XRD and TEM revealed MF8P_Ca salt formation and nano-layering at dentin. The MF8P_Ca salt was as stable as that of 10-MDP; MF8P was as hydrophobic as 10-MDP; a significantly higher bond strength was recorded for MF8P than for 10-MDP. In conclusion, MF8P chemically bonded to HAp. Despite its shorter size, MF8P possesses characteristics similar to those of 10-MDP, most likely to be associated with the strong chemical bond between fluorine and carbon. Since favorable bond strength to dentin was recorded, MF8P can be considered a good candidate functional monomer for bonding.

Mussel-inspired human gelatin nanocoating for creating biologically adhesive surfaces

Recombinant human gelatin was conjugated with dopamine using carbodiimide as a surface modifier. This dopamine-coupled human gelatin (D-rhG) was characterized by 1H-nuclear magnetic resonance, mass spectroscopy, and circular dichroism. D-rhG-coated surface properties were analyzed by physicochemical methods. Additionally, cell attachment and growth on the modified surfaces was assessed using human umbilical endothelial cells. Binding of gelatin onto titanium was significantly enhanced by dopamine conjugation. The thickness of the D-rhG coating depended on the treatment pH; thicker layers were formed at higher pH values, with a maximum thickness of 30 nm. D-rhG enhanced the binding of collagen-binding vascular endothelial growth factor and cell adhesion as compared with gelatin alone, even at the same surface concentration. The D-rhG surface modifier enhanced substrate binding by creating an adhesive nanointerface that increased specific protein binding and cell attachment.

Bone engineering by phosphorylated-pullulan and β-TCP composite

A multifunctional biomaterial with the capacity bond to hard tissues, such as bones and teeth, is a real need for medical and dental applications in tissue engineering and regenerative medicine. Recently, we created phosphorylated-pullulan (PPL), capable of binding to hydroxyapatite in bones and teeth. In the present study, we employed PPL as a novel biocompatible material for bone engineering. First, an in vitro evaluation of the mechanical properties of PPL demonstrated both PPL and PPL/β-TCP composites have higher shear bond strength than materials in current clinical use, including polymethylmethacrylate (PMMA) cement and α-tricalcium phosphate (TCP) cement, Biopex-R. Further, the compressive strength of PPL/β-TCP composite was significantly higher than Biopex-R. Next, in vivo osteoconductivity of PPL/β-TCP composite was investigated in a murine intramedular injection model. Bone formation was observed 5 weeks after injection of PPL/β-TCP composite, which was even more evident at 8 weeks; whereas, no bone formation was detected after injection of PPL alone. We then applied PPL/β-TCP composite to a rabbit ulnar bone defect model and observed bone formation comparable to that induced by Biopex-R. Implantation of PPL/β-TCP composite induced new bone formation at 4 weeks, which was remarkably evident at 8 weeks. In contrast, Biopex-R remained isolated from the surrounding bone at 8 weeks. In a pig vertebral bone defect model, defects treated with PPL/β-TCP composite were almost completely replaced by new bone; whereas, PPL alone failed to induce bone formation. Collectively, our results suggest PPL/β-TCP composite may be useful for bone engineering.

Phosphorylated Pullulan Bioadhesive for Regeneration and Reconstruction of Bone and Tooth

A biodegradable material that bonds to hard tissues such as bones and teeth is urgently needed for medical and dental applications. However, such materials are not available in today’s clinical practice of orthopedics and dentistry. Therefore, we synthesized biodegradable phosphorylated pullulan to develop a biomaterial that combines primary properties such as high biocompatibility, good bonding potential to hard tissue, high strength, biodegradability, and osteoconductivity. The pharmacopoeial polysaccharide pullulan was chemically functionalized with dihydrogen phosphate groups. Phosphorylated pullulan was formed network by adding calcium ion, making the composite less soluble in water. Adhesive force measurement revealed that adhesiveness of the pastes before setting can be controlled through optimization of additives. In addition, histological evaluation revealed that phosphorylated pullulan-based composite possesses high biocompatibility. These results indicate that phosphorylated pullulan can be used as a key material for regeneration and reconstruction of bone and tooth.

Electronic states of alkyl-radical-functionalized C20 fullerene using density functional theory

The structures and electronic states of alkyl-radical-functionalized C20 fullerenes (denoted by C20–R) have been investigated using density functional theory (DFT). The different alkyl radicals investigated were methyl, ethyl, propyl, and butyl radicals. The DFT calculation indicated that the alkyl radical binds to the carbon atom of C20 in the on-top site, thus forming a strong C–C single bond. The binding energies of the alkyl radicals to C20 were calculated to be 83.9–86.6 kcal/mol at the CAM-B3LYP/6-311G(d,p) level. The electronic states of the C20–R complex are discussed on the basis of the theoretical results.