Yasuaki Shibata

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.

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.

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

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-α (TNFα) 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 TNFα signaling plays some important roles in orthodontic tooth movement.

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP‐2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein‐2 (Bmp‐2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino‐terminal portion of SHH (rSHH‐N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3‐E1 cells. rSHH‐N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH‐N‐induced ALP activity in C3H10T1/2 and MC3T3‐E1 cells, indicating that BMPs are involved in SHH‐induced osteoblast differentiation. Simultaneous supplement with rSHH‐N and BMP‐2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH‐N for 6 h enhanced the response to BMP‐2 by increasing ALP activity in C3H10T1/2 and MC3T3‐E1 cells. Stimulatory effects of rSHH‐N and additive effects with rSHH‐N and BMP‐2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP‐2 (1 μg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r‐SHH‐N (1–5 μg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.

BMP-2 promotes differentiation of osteoblasts and chondroblasts in Runx2-deficient cell lines.

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD‐C6, from Runx2‐deficient mouse embryos. RD‐C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) or transduction of BMP‐2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD‐C6 cells. BMP‐2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD‐C6 cells with or without rhBMP‐2 treatment demonstrated that BMP‐2 upregulated 66 genes including 13 transcription‐related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD‐C6 cells, we transplanted RD‐C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP‐2. RD‐C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP‐2 induces Runx2‐deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2‐independent pathway, but it failed to induce these cells to differentiate into bone‐forming osteoblasts and mature chondrocytes. J. Cell. Physiol. 211: 728–735, 2007.

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

Abstract For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions.

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.

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.

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.