Effect of silicate incorporation on in vivo responses of α-tricalcium phosphate ceramics.

In addition to calcium phosphate-based ceramics, glass-based materials have been utilized as bone substitutes, and silicate in these materials has been suggested to contribute to their ability to stimulate bone repair. In this study, a silicate-containing α-tricalcium phosphate (α-TCP) ceramic was prepared using a wet chemical process. Porous granules composed of silicate-containing α-TCP, for which the starting composition had a molar ratio of 0.05 for Si/(P + Si), and silicate-free α-TCP were prepared and evaluated in vivo. When implanted into bone defects that were created in rat femurs, α-TCP ceramics either with or without silicate were biodegraded, generating a hybrid tissue composed of residual ceramic granules and newly formed bone, which had a tissue architecture similar to physiological trabecular structures, and aided regeneration of the bone defects. Supplementation with silicate significantly promoted osteogenesis and delayed biodegradation of α-TCP. These results suggest that silicate-containing α-TCP is advantageous for initial skeletal fixation and wound regeneration in bone repair.

A bone substitute with high affinity for vitamin D-binding protein--relationship with niche of osteoclasts.

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism.

Accelerated bone formation on photo-induced hydrophilic titanium implants: an experimental study in the dog mandible.

OBJECTIVES: The purpose of this study was to investigate the effect of photo-induced hydrophilic titanium dioxide (TiO2) on serum fibronectin (sFN) attachment, and further to evaluate initial osseointegration responses in the dog mandibles. MATERIALS AND METHODS: To apply the anatase TiO2 film, plasma source ion implantation (PSII) method followed by annealing was employed for the titanium disks and implants, which were then illuminated with UV-A for 24 h for the experimental groups. Non-deposited titanium disks and implants were prepared for the control group. Surface characterization was performed using the interferometer and contact angle analyzer. The attachments of sFN were evaluated using fluorescence emission analysis. Thereafter both groups of implants were placed in the mandible of six beagle dogs. Bone response was investigated with histological and histomorphometrical analyses after periods of 2 and 4 weeks. RESULTS: The experimental groups exhibited strong hydrophilicity under UV-A illumination and showed significant improvement in sFN attachment. And further, the experimental implants enhanced the bone formation with the bone-to-implant contact of 42.7% after 2 weeks of healing (control: 28.4%). CONCLUSIONS: The combined applications of plasma fibronectin and PSII to produce hydrophilic titanium surfaces could accelerate early osseointegration.

Pathological differences in the bone healing processes between tooth extraction socket and femoral fracture.

Despite various reports on the bone healing processes of tooth extraction socket and long bone fracture, the differences of pathological changes during these healing processes remain elusive. This study aims to elucidate the underlying mechanisms behind the pathophysiology of bone regeneration between the tooth extraction socket and femoral fractures through a comparative study. Eight-week-old male mice were used in the experiments. The maxillary first molar was extracted, and intramedullary nailing femoral fracture (semistabilized fracture repair) was performed in the femur. Pathological changes in these bone injuries were investigated by micro-CT, histology, immunohistochemistry, and RT-PCR until day 7 post operation. Pathological changes in drill hole injury created in cortical bone of femur were also examined. Micro-CT analyses revealed increases in mineralized tissues in both the tooth extraction socket and femoral fracture. Histological examinations revealed that tooth socket was repaired by intramembranous ossification, and intramedullary nailing femoral fracture was healed by endochondral ossification. Immunohistochemical investigation revealed that tooth socket healing associated with Sp7-positive cells but not Sox9, aggrecan, and type II collagen, while femoral fracture models exhibited positive signals for all antibodies. RT-PCR analyses revealed the expression of Sp7, Col1a1, and Col2a1 in tooth socket healing, and the expression of Sp7, Col1a1, Runx2, Sox9, Acan, Col2a1, and Col10a1 in intramedullary nailing femoral fracture. Drill hole injury was repaired primarily by intramembranous ossification when the periosteum was removed before making the hole. The present study demonstrated that the absence of cartilage appearance during tooth extraction socket healing indicates it as distinctly different pathological features from the healing processes of semistabilized femoral fracture. This study contributes to the understanding of the molecular and cellular characteristics of bone healing among the different sites of bone injury.

The slow resorption with replacement by bone of a hydrothermally synthesized pure calcium-deficient hydroxyapatite.

A newly developed calcium-deficient hydroxyapatite composed of rod-shaped particles synthesized by the hydrothermal method (HHA) and stoichiometric hydroxyapatite (SHA) synthesized by the sintering method was used for in vivo implantation and in vitro culture systems to compare these biological responses. In the rabbit femur, implanted HHA was slowly resorbed and about 80% of the implant remained 24 weeks after implantation; however, up to 72 weeks after implantation, most of the implanted HHA was resorbed. The implanted SHA was unresorbed throughout the experimental period, but degradation by the invasion of newly formed bone was seen at 72 weeks after implantation. Bone histomorphometry showed that the volume of newly formed bone and the number of osteoclasts in the implanted region were significantly higher in HHA than in SHA 24 weeks after implantation. In vitro culture of C2C12 cells with the induction of osteoblastic phenotypes using recombinant bone morphogenetic protein-2 showed similar cell density and the induction of alkaline phosphatase activity between the cells on HHA and SHA discs. In vitro osteoclastogenesis of HHA and SHA discs using bone marrow macrophages and recombinant receptor activator of nuclear factor-kappaB ligand showed higher TRAP activity of osteoclasts cultured on HHA discs. These results showed that slow biodegradability did not always correlate to final replaceability in bone tissue, and suggested that the activity of osteoclasts correlated to the bone-forming activity of osteoblasts.

Photo-induced hydrophilicity enhances initial cell behavior and early bone apposition.

OBJECTIVE: The anatase form of titanium dioxide (TiO(2)) exhibits photo-induced hydrophilicity when it is irradiated with ultraviolet (UV) light. In the present study, the effect of photo-induced hydrophilicity on initial cell behavior and bone formation was evaluated. MATERIALS AND METHODS: Plasma source ion implantation method and post-annealing were employed for coating the anatase form of TiO(2) to the surface of the titanium disk and implant. Half of the disks and implants were illuminated with UV for 24 h beforehand, whereas the other halves were blinded and used as controls. Photo-induced hydrophilicity was confirmed by a static wettability assay. The effects of this hydrophilicity on cell behavior were evaluated by means of cell attachment, proliferation and morphology using pluripotent mesenchymal precursor C2C12 cells. Thereafter, bone formation around the hydrophilic implant inserted in the rabbit tibia was confirmed histomorphometrically. RESULTS: The water contact angle of the photo-induced hydrophilic disk decreased markedly from 43.5 degrees to 0.5 degree. Cell attachment and proliferation on this hydrophilic disk showed significant improvement. The cell morphology on this hydrophilic disk was extremely flattened, with an elongation of the lamellipodia, whereas a round/spherical morphology was observed on the control disk. The photo-induced hydrophilic implant enhanced the bone formation with the bone-to-metal contact of 28.2% after 2 weeks of healing (control: 17.97%). CONCLUSION: The photo-induced hydrophilic surface used in the current study improves the initial cell reactions and enhances early bone apposition to the implant.

Enhanced initial cell responses to chemically modified anodized titanium.

BACKGROUND: Previously, we reported that anodized porous titanium implants have photocatalytic hydrophilicity. However, this effect was not always sufficient for the significant improvement of bone apposition. PURPOSE: The purpose of this study was to improve the photocatalytic properties of porous titanium implants by the fluoride modification of the anodized titanium dioxide (TiO(2)), and to investigate the initial cell response to it. MATERIALS AND METHODS: The ideal concentration of ammonium hydrogen fluoride (NH(4)F-HF(2)) used in this study was determined by a static water contact angle assay. The ideal concentration of NH(4)F-HF(2) was 0.175%, and experimental disks were treated with this concentration. A pluripotent mesenchymal cell line, C2C12, was cultured on the disks in order to investigate cell attachment, morphology, and proliferation. RESULTS: Cell attachment after 30 minutes of culturing was significantly higher for the ultraviolet-irradiated, fluoride-modified anodized TiO(2) (p < .05), and the simultaneous scanning electron microscope observation showed a rather flattened and extended cell morphology. The proliferation rate after 24 hours was also significantly higher for the fluoride-modified anodized TiO(2). CONCLUSION: Fluoride chemical modification enhances the hydrophilic property of the anodized TiO(2) and improves the initial cell response to it.

The effect of the microstructure of beta-tricalcium phosphate on the metabolism of subsequently formed bone tissue.

The response of bone cells to a newly developed porous beta-tricalcium phosphate composed of rod-shaped particles (RSbeta-TCP), beta-TCP composed of conventional non-rod-shaped particles (Cbeta-TCP), and hydroxyapatite (HA) was analyzed using in vivo implantation and in vitro osteoclastogenesis systems. Implantation of the materials into the rabbit femur showed that RSbeta-TCP and Cbeta-TCP were bioresorbable, but HA was not. Up to 12 weeks after the implantation, bioresorption of RSbeta-TCP and Cbeta-TCP accompanied by the formation of new bone occurred satisfactorily. At 24 weeks post-implantation, most of the RSbeta-TCP had been absorbed, and active osteogenesis was preserved in the region. However, in the specimens implanted with Cbeta-TCP, the amount of not only the implanted Cbeta-TCP but also the newly formed bone tissue decreased, and bone marrow dominated the region. The implanted HA was unbioresorbable throughout the experimental period. When osteoclasts were generated on RSbeta-TCP, Cbeta-TCP, or HA disks, apparent resorption lacunae were formed on the RSbeta-TCP and Cbeta-TCP, but not HA disks. Quantitation of the calcium concentration in the culture media showed an earlier and more constant release of calcium from RSbeta-TCP than Cbeta-TCP. These results showed that the microstructure of beta-TCP affects the activity of bone cells and subsequent bone replacement.

Effects of cleaning methods for custom abutment surfaces on gene expression of human gingival fibroblasts.

The aim of this study was to develop an effective method for cleaning implant abutments made by computer-aided design and computer-aided manufacturing techniques and to investigate the effect of decontamination in vitro. Briefly, a newly developed reagent (PK) and/or vacuum plasma (Plasma) were used to clean the surfaces of zirconia disks, and the effects of this decontamination were evaluated by X-ray photoelectron spectroscopy. Human gingival fibroblasts (HGFs) were cultured on sample disks for 6, 24, and 48 h. We evaluated cell attachment and gene expression of the acute inflammatory cytokines interleukin-6 and vascular endothelial growth factor A, and type 1 collagen. In the PK and PK+Plasma groups, surface contaminants were reduced by washing. In addition, HGF attachments was increased in the PK and PK+Plasma groups. Gene expressions of interleukin-6 and vascular endothelial growth factor A were lower at 6 h. Gene expression of type 1 collagen was increased at all time points after seeding. These results suggest that decontamination of implant abutment surfaces is important in initial HGF attachment and may improve the biological seal of peri-implant soft tissue.

Coexpression of ang1 and tie2 in odontoblasts of mouse developing and mature teeth-a new insight into dentinogenesis.

Angiopoietin-1 regulates vascular angiogenesis and stabilization, and is reported to promote bone formation by facilitating angiogenesis. To estimate the role of Ang1 in odontogenesis, we explored the distribution of Ang1 and the receptor, Tie2 in the mouse developing and mature first molar of the mandible. At embryonic day 18, when differentiation of odontoblasts begins, immunosignals for Ang1 were intensely detected in the basement membrane and the distal side, which faced the basement membrane of odontoblasts. In situ hybridization revealed that Ang1 was expressed in odontoblasts and ameloblasts facing the basement membrane. Tie2 was localized in the distal side of odontoblasts. After birth, Ang1 was detected in the predentin, whereas both Ang1 and Tie2 were colocalized in odontoblasts and odontoblast processes. These distributions were retained up to 8 weeks. In contrast to odontoblasts, ameloblasts, cementoblasts and osteoblasts expressed Ang1 but did not express Tie2. Colocalization of Ang1 and Tie2 in odontoblasts and selective expression of Tie2 in odontoblasts among cells responsible for calcified tissue formation suggested the involvement of autocrine signals of Ang1-Tie2 in dentinogenesis.

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts.

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, beta-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.

Experimental model of tooth movement by orthodontic force in mice and its application to tumor necrosis factor receptor-deficient mice.

Orthodontic tooth movement is achieved by mechanical loading; however, the biological mechanism involved in this process is not clearly understood owing to the lack of a suitable experimental model. In the present study, we established an orthodontic tooth movement model in mice using a Ni-Ti closed coil spring that was inserted between the upper incisors and the upper first molar. Histological examination demonstrated that the orthodontic force moved the first upper molar mesially without necrosis of the periodontium during tooth movement. The number of TRAP-positive osteoclasts on the pressure side significantly increased in a time-dependent manner. Quantitative real time-based reverse transcription-polymerase chain reaction analysis demonstrated increased levels of mRNA for cathepsin K. Immunohistochemical staining revealed the expression of tumor necrosis factor-alpha (TNFalpha) in periodontium on the pressure side of the first molar during orthodontic tooth movement. When this tooth movement system was applied to TNF type 1 receptor-deficient mice and TNF type 2 receptor-deficient mice, tooth movement observed in TNF type 2 receptor-deficient mice was smaller than that in the wild-type mice and TNF type 1 receptor-deficient mice. The number of TRAP-positive osteoclasts on the pressure side was significantly small in TNF type 2 receptor-deficient mice compared with that in TNF type 1 receptor-deficient mice on day 6 after application of the appliance. The present study indicates that TNFalpha signaling plays some important roles in orthodontic tooth movement.

Expressão de TGFβ1 mRNA nas fases iniciais de expansão da sutura palatina mediana / The expression of TGFβ1 mRNA in the early stage of the midpalatal suture cartilage expansion

INTRODUÇÃO: a expansão da maxila induz a formação de novo osso na sutura palatina mediana por um processo de proliferação e diferenciação celular. A força de expansão pode estimular, nas células progenitoras, a produção de citocinas com atividade osteoindutiva, tais como o transforming growth factor β1(TGFβ1). OBJETIVOS: o principal objetivo deste estudo foi determinar a função dessa citocina nos estágios iniciais de expansão da sutura palatina mediana. MÉTODOS: um aparelho ortodôntico foi instalado entre os molares superiores direito e esquerdo de ratos com 4 semanas de idade. A força de expansão inicial foi de 50g. Os grupos controle e experimental foram sacrificados nos dias 0, 2 e 5. Cortes bucais de 6µm foram obtidos e sujeitos à técnica de hibridização in-situ. RESULTADOS: dois dias após a aplicação de força, as células osteocondroprogenitoras, distribuídas no lado interno do tecido cartilaginoso, exibiram altos níveis de transcrição de transforming growth factor β1. No dia 5, o nível de transcrição de TGFβ1 foi observado nos osteócitos e nas células osteoblásticas, na superfície do novo osso. A atividade osteoblástica foi confirmada por meio de um estudo imunohistoquímico utilizando-se Osteocalcina-Pro (OC-Pro). CONCLUSÕES: os dados sugerem que a expansão da sutura palatina induz a diferenciação de células osteocondroprogenitoras em osteoblastos, estimuladas pela produção de citocinas.

INTRODUCTION: The application of an orthodontic expansion force induces bone formation at the midpalatal suture because of cell proliferation and differentiation. Expansion forces may stimulate the production of osteoinductive cytokines, such as transforming growth factor β1 (TGFβ1), in the progenitor cells. OBJECTIVES: This study determined the role of TGFβ1 in the early stage of midpalatal suture cartilage expansion. METHODS: A rectangular orthodontic appliance was placed between the right and left upper molars of 4-week-old rats. The initial expansion force was 50 g. Animals in the control and experimental groups were sacrified on days 0, 2, and 5 and 6 µmm thick sections were prepared for an in situ hybridization technique. RESULTS: Two days after the application of force, prechondroblastic and undifferentiated mesenchymal cells distributed along the inner side of the cartilaginous tissue had high levels of TGFβ1 transcription. On day 5, the TGFβ1 transcription was found in osteocytes and osteoblastic cells on the surface of newly formed bone. Immunohistochemistry using Osteocalcin-Pro (OC-Pro) confirmed osteoblastic activity. Conclusions: Results suggest that the expansion of midpalatal suture cartilage induces differentiation of osteochondroprogenitor cells into osteoblasts after stimulation by cytokine production.

Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ.

Rodents have brownish-yellow incisors whose colour represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region-leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2-deficient mice show decolourization of the incisors. While incisors of wild-type mice were brownish yellow, incisors of Nrf2-deficient mice were greyish white in colour. Micro X-ray imaging analysis revealed that the iron content in Nrf2-deficient mouse incisors were significantly decreased compared to that of wild-type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2-deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2-null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2-deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition on to the enamel surface.