An oriented hydroxyapatite film with arrayed plate-like particles enhance chondrogenic differentiation of ATDC5 cells.

Osteochondral defects of articular cartilage cannot regenerate spontaneously. For its surgical treatment, advancements in cartilage tissue engineering have particularly focused on subchondral bone lesions that tend to delay healing. Therefore, it is important to understand interactions between subchondral bone and chondrocytes. This study aimed to investigate the behavior of chondrogenic ATDC5 cells on oriented hydroxyapatite (HAp) films that mimic bone surfaces. HAp nanoparticles prepared herein were needle like and plate like. HAp films were formed through self-organization of the nanoparticles and had 2D structures regularly arrayed with the particles. Both films prominently comprised a-plane orientation surfaces but differed in the degree of hydrophilicity because of the patterns of particle self-assembly. ATDC5 cells cultured on the HAp film with plate-like particles could adhered in a shorter period but could not spread. The adhesive force of cells was weaker with the hydrophilic surface than with other surfaces, as determined using a trypsin-based cell detachment assay. In addition, ATDC5 cells displayed enhanced proliferation and chondrogenic differentiation. Our results suggest that the oriented HAp film formed using plate-like particles provided chondrogenic cells with a desired scaffold as that of subchondral bone to increase cell proliferation and differentiation.

Plate-like hydroxyapatite synthesized from dodecanedioic acid enhances chondrogenic cell proliferation.

BACKGROUND: The scaffold for head and neck reconstruction needs mechanical strength to maintain specific forms. Hydroxyapatite (HA) enhances the mechanical strength of hydrogel and is routinely used for cartilage regeneration. However, there is a demand for hydroxyapatite that controls chondrogenic cell behavior. OBJECTIVE: Our aim was to regulate HA morphology through a hydrothermal process using organic acid and enhance chondrocyte proliferation and differentiation using shaped-regulated HA. METHODS: HA was synthesized from dodecanedioic acid (DD:HA) and oleic acid (OA:HA) by a hydrothermal method and then coated onto glass plates. Surface properties of the samples were compared by various techniques. Surface roughness and contact angles were calculated. Proliferation and differentiation of chondrogenic cells were measured by MTT assays and Alcian Blue staining, respectively, after various incubation periods. RESULTS: The morphological structures of DD:HA and OA:HA were different; however, the crystallinity and chemical structures were similar. Surface roughness and hydrophilic behavior were higher on DD:HA. DD:HA enhanced chondrogenic cell proliferation over time. The differentiation of ATDC5 cells was also increased on the DD:HA surface compared with those in other groups. CONCLUSIONS: DD:HA enhanced cell viability to a greater extent than OA:HA did, indicating its excellent potential as an inorganic material compatible with chondrocyte regeneration.

Transparent non-cubic laser ceramics with fine microstructure.

Transparent polycrystalline ceramics with cubic crystal structure have played important roles in a wide variety of solid-state laser applications, whereas for non-cubic structures, single crystal only has been used. For further progress in optical technologies, effective materials beyond the current limitations are necessary. Here we report a new type of non-cubic ceramic laser material that overturns conventional common sense. It is hexagonal Nd-doped fluorapatite (Nd:FAP) ceramics with an optical quality comparable to single crystal while having random crystal orientation. It is composed of ultrafine grains with a loss coefficient of 0.18 cm-1 at a lasing wavelength of 1063 nm, and its laser oscillation was demonstrated. This is the first verification of lasing in randomly oriented non-cubic ceramics. Laser oscillation in the non-cubic ceramics was realized through both advanced liquid-phase nano-powder synthesis technology and highly controlled pulsed-current sintering techniques. Our findings should open new avenues for future solid-state laser and optical applications.

The effect of glucose modification of hydroxyapatite nanoparticles on gene delivery.

Surface modification techniques have been employed for the use of biocompatible and bioresorbable hydroxyapatite (HAp) nanoparticles in cell biology and medicine for the delivery of bioactive molecules. We demonstrated the effects of glucose modification of HAp (GlcHAp) on the transfection efficiency in endothelial cells. After preparing homogeneous HAp nanoparticles with a microemulsion technique, the particles with or without glucose modification and plasmid DNA (pDNA) complexes were transfected into endothelial cells. The transfection efficiency of GlcHAp/pDNA was higher than that of HAp/pDNA. To elucidate the mechanism underlying the improvement in the transfection efficiency following glucose modification, the uptake route into the cells and the inhibition of DNA degradation were investigated. GlcHAp/pDNA enhanced the transfection efficiency after interacting with the glucose transporter 1, as observed by the selective inhibitor assay. In addition, GlcHAp/pDNA was more stable than HAp/pDNA in the DNA degradation assay. Our results suggest that the glucose modification could promote the uptake of HAp nanoparticles by cells and protect the internalized DNA; properties essential for non-viral transfection carriers. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 61-66, 2019.

Regulation of periodontal ligament-derived cells by type III collagen-coated hydroxyapatite.

BACKGROUND: The periodontal ligament (PDL), which maintains homeostasis in the periodontium, is a group of specialized connective tissue fibers attached to both the cementum and alveolar bone. Regeneration of periodontium with PDL cells has been investigated, and the chemical and molecular structures of scaffolds control the adhesion and differentiation of cells. Therefore, the development of adequate materials for PDL-derived cells is essential to regenerate the periodontium. OBJECTIVE: We evaluated the suitable passage time for PDL-derived cells and investigated the behaviors of PDL-derived cells grown on hydroxyapatite (HAp) scaffolds coated with type I and type III collagen. METHODS: PDL-derived cells were isolated with enzyme from the upper molars of male Wister rats. After characterization of HAp, type I collagen, and type III collagen, PDL-derived cells at passage 2 were seeded onto collagen-coated HAp. Cell adhesion, proliferative potential, and osteoconductivity were analyzed with immunostaining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Alizarin S staining, and real-time polymerase chain reaction. RESULTS: Type I and III collagens were successfully coated on HAp. Gene expression analysis revealed that passage 2 was suitable for maintaining differentiation potential. Proliferative potential and cell adhesion were significantly higher on type III collagen than on HAp alone or type I collagen. In contrast, the osteoconductivity of type III collagen was significantly lower than those of HAp and type I collagen. CONCLUSION: PDL-derived cells on type I collagen differentiated into osteogenic cells and formed hard tissues. However, type III collagen enhanced the adhesion of PDL-derived cells and inhibited mineralization.

Ag nanoparticle-coated zirconia for antibacterial prosthesis.

Bacterial adhesion to dental materials is a major cause of caries and periodontitis, necessitating the development of compounds such as yttria-stabilized zirconia (YSZ) and silver nanoparticles (AgNPs), which are widely employed in medicine due to their high antimicrobial activity and low cytotoxicity. The main goal of this study is the synthesis of the broad antimicrobial activity of AgNP-coated YSZ with facile methods. The bactericidal AgNPs were immobilized on the surface of YSZ and tested for bactericidal activity against Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Aggregatibacter actinomycetemcomitans based on ISO 22196:2007. The loading of AgNPs was optimized by culturing mouse fibroblast cells on AgNP-coated YSZ with cell viability test based on ISO 10993-5. In addition, the silver release profile of AgNP-coated YSZ in artificial saliva was determined using an accelerated aging test. Antibacterial activity, and cell viability test revealed optimum performance with no cytotoxicity at a level of 32 µg/cm2. Accelerated aging test showed that the AgNP-coated surface was extremely stable, exhibiting a total silver leaching level of only 1% and confirming the effectiveness of this coating method for retaining AgNPs while exerting an antibacterial effect against oral pathogens. This finding also implies that the bactericidal action of AgNP-coated YSZ is not mediated by the released Ag ions, but rather corresponds to contact killing. ABBREVIATIONS: Yttria-stabilized zirconia, YSZ; silver nanoparticles, AgNPs; field emission scanning electron microscopy, FE-SEM; X-ray photoelectron spectroscopy, XPS; grazing incidence X-ray diffraction, GIXRD; ultraviolet-visible, UV-vis; inductively coupled plasma atomic emission spectrometry, ICP-AES.

Effects of controlled micro-/nanosurfaces on osteoblast proliferation.

Micro-/nanostructured implant surfaces mimicking natural bone architecture are expected to have positive effects on osteoblast proliferation. In this study, to clarify the optimal microstructure scale on micro-/nano-titanium surfaces, highly controlled microstructure surfaces were fabricated with a titania nanotube oxide layer by sandblast treatment and electrochemical anodization. Average surface roughness (Ra ) and mean width of the profile elements (RSm ) increased in proportion to the size of the blasting media. The crystalline structure and wettability were similar for all samples after anodizing and annealing. MG63 osteoblast-like osteosarcoma cells showed reduced spreading area on smaller microstructure surfaces after incubation for 24 h. Cell proliferation assays, including cell counting and cell cycle analyses, showed that micro-/nanosurfaces with smaller microstructures (Ra = 0.6 µm, RSm = 11.1 µm) had the most positive effect. This result indicated that smaller microstructures of hierarchical surfaces enhanced cell proliferation, despite similar chemical compositions and wettabilities. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2589-2596, 2017.

Synthesis and enhanced bone regeneration of carbonate substituted octacalcium phosphate.

Using a wet method, we have synthesized octacalcium phosphate carbonate, in which HPO42- in octacalcium phosphate is replaced with CO32-. The physical, crystal, and chemical properties of this new material were compared to octacalcium phosphate, Ca-deficient hydroxyapatite, and Ca-deficient carbonate apatite using X-ray diffraction, Fourier-transform infrared spectroscopy, inductively coupled plasma spectroscopy, and scanning electron microscopy. Surface roughness and morphology were also characterized, along with the ability to support proliferation and differentiation of MG63 cells, as measured by MTT and alkaline phosphatase assay. We found that octacalcium phosphate carbonate enhanced osteoblast proliferation more strongly than all other materials tested. Similarly, Ca-deficient carbonate apatite, a hydrolysate of octacalcium phosphate carbonate, stimulated osteoblast differentiation to a better extent than Ca-deficient hydroxyapatite, a carbonate-free hydrolysate of octacalcium phosphate. These results indicate that octacalcium phosphate carbonate has good biocompatibility and osteoconduction, and incorporation of carbonate into octacalcium phosphate and apatite enhances bone regeneration.

Influences of osteoarthritis and osteoporosis on the electrical properties of human bones as in vivo electrets produced due to Wolff's law.

We characterized the electrical properties of living bone obtained from patients who had undergone total hip arthroplasty (THA) or hemiarthroplasty by means of analysis of the electrically polarized and nonpolarized bone specimens, and we discussed the role of an organic and inorganic matrix of human bone in bone piezoelectricity.We used human femoral neck bone that was harvested during THA for advanced osteoarthritis of the hip joint (OA group) and hemiarthroplasty for femoral neck fracture (FNF group). The specimens were scanned to evaluate the cancellous bone structures using micro-computed tomography, and we quantified the carbonic acid by attenuated total reflection (ATR) spectra to estimate carbonate apatite. The stored electrical charge in the electrically polarized and nonpolarized bone specimens were calculated using thermally stimulated depolarized current (TSDC) measurements.Each TSDC curve in the groups had peaks at 100°C, 300°C and 500°C, which may be attributed to collagen, carbonate apatite and hydroxyapatite, respectively. It is suggested that organic substances are more effectively electrically polarized than apatite minerals by the polarization at room temperature and that the stored charge in living bone may be affected not only by total bone mass but also by bone quality, including 3-dimensional structure and structural component.

Morphological and functional changes in RAW264 macrophage-like cells in response to a hydrated layer of carbonate-substituted hydroxyapatite.

Synthetic hydroxyapatite (HAp) is used clinically as a material for bone prostheses owing to its good bone-bonding ability; however, it does not contribute to bone remodeling. Carbonate-substituted hydroxyapatite (CAp) has greater bioresorption capacity than HAp while having similar bone-bonding potential, and is therefore considered as a next promising material for bone prostheses. However, the effects of the CAp instability on inflammatory and immune responses are unknown in detail. Here, we show that the surface layer of CAp is more hydrated than that of HAp and induces changes in the shape and function of macrophage-like cells. HAp and CAp were synthesized by wet method and molded into disks. The carbonate content of CAp disks was 6.2% as determined by Fourier transform (FT) infrared spectral analysis. Diffuse reflectance infrared FT analysis confirmed that physisorbed water and surface hydroxyl groups (OH- ) were increased whereas structural OH- was decreased on the CAp as compared to the HAp surface. The degree of hydroxylation in CAp was comparable to that in bone-apatite structures, and the CAp surface exhibited greater hydrophilicity and solubility than HAp. We investigated immune responses to these materials by culturing RAW264 cells (macrophage precursors) on their surfaces. Cell spreading on the CAp disk was suppressed and the secretion level of inflammatory cytokines was reduced as compared to cells grown on HAp. These results indicate that the greater surface hydration of CAp surface can attenuate adverse inflammatory responses to implanted bone prostheses composed of this material. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1063-1070, 2017.

The role of the collaborative functions of the composite structure of organic and inorganic constituents and their influence on the electrical properties of human bone.

BACKGROUND: The electrical potential, which is generated in bone by collagen displacement, has been well documented. However, the role of mineral crystals in bone piezoelectricity has not yet been elucidated. OBJECTIVE: We examined the mechanism that the composite structure of organic and inorganic constituents and their collaborative functions play an important role in the electrical properties of human bone. METHODS: The electrical potential and bone structure were evaluated using thermally stimulated depolarized current (TSDC) and micro computed tomography, respectively. After electrical polarization of bone specimens, the stored electrical charge was calculated using TSDC measurements. The CO3/PO4 peak ratio was calculated using attenuated total reflection to compare the content of carbonate ion in the bone specimens. RESULTS: The TSDC curve contained 3 peaks at 100, 300 and 500°C, which were classified into 4 patterns. The CO3/PO4 peak ratio positively correlated with the stored charges at approximately 300°C in the polarized bone. There was a positive correlation between the stored bone charge and the bone mineral density only. CONCLUSIONS: It is suggested that the peak at 300°C is attributed to carbonate apatite and the total bone mass of human bone, not the three-dimensional structure, affects the stored charge.

Suppression effects of dental glass-ceramics with polarization-induced highly dense surface charges against bacterial adhesion.

This study investigated the surface characteristics and antibacterial ability capacity of surface-improved dental glass-ceramics by an electrical polarization process. Commercially available dental glass-ceramic materials were electrically polarized to induce surface charges in a direct current field by heating. The surface morphology, chemical composition, crystal structure, and surface free energy (SFE) were evaluated using scanning electron microscopy, energy dispersive X-ray spectrometry, X-ray diffraction, and water droplet methods, respectively. The antibacterial capacity was assessed by a bacterial adhesion test using Streptococcus mutans. Although the surface morphology, chemical composition, and crystal structure were not affected by electrical polarization, the polar component and total SFE were enhanced. After 24 h incubation at 37ºC, bacterial adhesion to the polarized samples was inhibited. The electrical polarization method may confer antibacterial properties on prosthetic devices, such as porcelain fused to metal crowns or all ceramic restorations, without any additional bactericidal agents.

Hierarchical periodic micro/nano-structures on nitinol and their influence on oriented endothelialization and anti-thrombosis.

The applications of hierarchical micro/nano-structures, which possess properties of two-scale roughness, have been studied in various fields. In this study, hierarchical periodic micro/nano-structures were fabricated on nitinol, an equiatomic Ni-Ti alloy, using a femtosecond laser for the surface modification of intravascular stents. By controlling the laser fluence, two types of surfaces were developed: periodic nano- and micro/nano-structures. Evaluation of water contact angles indicated that the nano-surface was hydrophilic and the micro/nano-surface was hydrophobic. Endothelial cells aligned along the nano-structures on both surfaces, whereas platelets failed to adhere to the micro/nano-surface. Decorrelation between the responses of the two cell types and the results of water contact angle analysis were a result of the pinning effect. This is the first study to show the applicability of hierarchical periodic micro/nano-structures for surface modification of nitinol.

Enhanced osteoconductivity of titanium implant by polarization-induced surface charges.

This study introduces the application of method for electrically polarizing titanium implants coated with anatase TiO2 using microarc oxidation. It also describes the features of the electrically polarized titanium implants, on which surface charges are generated by the dipole moment of the TiO2 , and describes how the surface charges affect the implants' in vivo bone-implant integration capability. A comprehensive assessment using biomechanical, histomorphological, and radiographic analyses in a rabbit model was performed on polarized and nonpolarized implants. The electrically polarized surfaces accelerated the establishment of implant biomechanical fixation, compared with the nonpolarized surfaces. The percentage of the bone-implant contact ratio was higher using polarized implants than using nonpolarized implants. In contrast, the bone volume around the implants was not affected by polarization. Thus, using the polarized implant, this study identified that controlled surface charges have a significant effect on the properties of titanium implants. The application of the electrical polarization process and the polarization-enhanced osteoinductivity, which resulted in greater bone-implant integration, was clearly demonstrated.