Global changes in epigenomes during mouse spermatogenesis: possible relation to germ cell apoptosis.

Mammalian spermatogenesis is characterized by disproportionate germ cell apoptosis. The high frequency of apoptosis is considered a safety mechanism that serves to avoid unfavorable transmission of paternal aberrant genetic information to the offspring as well as elimination mechanism for removal of overproduced immature or damaged spermatogenic cells. The molecular mechanisms involved in the induction of germ cell apoptosis include both intrinsic mitochondrial Bcl-2/Bax and extrinsic Fas/FasL pathways. However, little is known about the nuclear trigger of those systems. Recent studies indicate that epigenomes are essential in the regulation of gene expression through remodeling of the chromatin structure, and are genome-like transmission materials that reflect the effects of various environmental factors. In spermatogenesis, epigenetic errors can act as the trigger for elimination of germ cells with abnormal chromatin structure, abnormal gene expression and/or morphological defects (disordered differentiation). In this review, we focus on the relationship between global changes in epigenetic parameters and germ cell apoptosis in mice and other mammals.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

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.

In vivo over-expression of KGF mimic human middle ear cholesteatoma.

We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.

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.

CCN3 protein participates in bone regeneration as an inhibitory factor.

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.

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.

Transcriptome Profiling of Anhidrotic Eccrine Sweat Glands Reveals that Olfactory Receptors on Eccrine Sweat Glands Regulate Perspiration in a Ligand-Dependent Manner.

Sweat maintains systemic homeostasis in humans. Although sweating disorders may cause multifaceted health problems, therapeutic options for sweat disorders have not yet been established. To gain new insight into the mechanism underlying the regulation of perspiration, we compared eccrine sweat gland transcriptomes from hidrotic and anhidrotic lesions from patients with anhidrosis and found out that olfactory receptors were expressed differentially in anhidrotic and hidrotic eccrine sweat glands. We then confirmed OR51A7 and OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase-TGFα shedding assay revealed that ß-ionone activates G-proteins through OR51A7 or OR51E2. The effect of topically applied ß-ionone on sweating was examined with the quantitative sudomotor axon reflex test, which showed that responses to ß-ionone differed between sexes. Topical ß-ionone attenuated female sweating and augmented male sweating. Taken together, this study suggests that olfactory receptors expressed in eccrine sweat glands may regulate sweating in response to odorous ligands on the basis of sex. These unexpected results indicate that olfactory receptors may modulate sweating and that olfactory receptor modulators may contribute to the management of sweat disorders.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1 week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

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.

Pathogenesis of middle ear cholesteatoma: a new model of experimentally induced cholesteatoma in Mongolian gerbils.

Middle ear cholesteatoma is characterized by enhanced proliferation of epithelial cells with aberrant morphological characteristics. To investigate the origin of the cholesteatoma cells, we analyzed spontaneously occurring cholesteatomas associated with a new transplantation model in Mongolian gerbils (gerbils). Cholesteatomas were induced in gerbils with a transplanted tympanic membrane by using the external auditory canal (EAC) ligation method. After the pars flaccida of the tympanic membranes were completely removed from male gerbils, corresponding portions of tympanic membranes of female gerbils were transplanted to the area of defect, and then we ligated the EAC (hybrid-model group). As a control group, the EAC of normal male and female gerbils was ligated without myringoplasty. In all ears of each group, the induced cholesteatomas were seen. In situ PCR was then performed to detect the mouse X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene on the paraffin sections. One pgk-1 spot in the epithelial nuclei was detected in male cholesteatoma, and two pgk-1 spots were detected in female cholesteatoma, respectively. On the other hand, in the hybrid-model group, we detected not only one but also two pgk-1 spots in the epithelial nuclei of cholesteatoma. These results strengthened the evidence that the origin of epithelial cells in cholesteatoma is the tympanic membrane in this model, but not the residential middle ear epithelial cells or the skin of the EAC.

Nuclear Expression of Pygo2 Correlates with Poorly Differentiated State Involving c-Myc, PCNA and Bcl9 in Myanmar Hepatocellular Carcinoma.

In Myanmar, hepatocellular carcinoma (HCC) is commonly seen in young adult and associated with poor prognosis, while the molecular mechanisms that characterize HCC in Myanmar are unknown. As co-activation of Wnt/ß-catenin signaling and c-Myc (Myc) are reported to associate with malignancy of HCC, we immunohistochemically investigated the expression of Pygo2 and Bcl9, the co-activators of the Wnt/ß-catenin signaling, Myc and PCNA in 60 cases of Myanmar HCC. Pygo2 expression was confirmed by in situ hybridization. The signal intensity was measured by image analyzer and then statistically analyzed. As a result, the expression of Pygo2 was significantly higher in HCC compared to normal liver tissue and the nuclear signal was the most intense in poorly differentiated HCC. Cytoplasmic Bcl9 was expressed in the normal liver tissue but decreased in HCC with the progression of histopathological grade. Myc was significantly higher in poorly differentiated HCC, whereas PCNA labeling index increased with the progression of histopathological grade. Nuclear Pygo2 showed strong correlation with nuclear Myc (P < 0.01) and PCNA (P < 0.001), and inversely correlated with cytoplasmic Bcl9 (P < 0.01). Our results suggested Wnt/ß-catenin and Myc signaling is commonly activated in Myanmar HCC and that the correlative upregulation of nuclear Pygo2 and Myc characterizes the malignant features of HCC in Myanmar.

NS5A-ISGylation via Lysine 26 Has a Critical Role for Efficient Propagation of Hepatitis C Virus Genotype 2a.

We previously reported that hepatitis C virus (HCV) NS5A (1b, Con1) protein accepts covalent ISG15 conjugation at specific lysine (Lys) residues (K44, K68, K166, K215 and K308), exhibiting proviral effects on HCV RNA replication. Here we investigated a role of NS5A-ISGylation via Lys residues in HCV propagation using HCV infectious clone. The alignment of amino acid sequences revealed that 5 Lys residues (K20, K26, K44, K139, and K166) of the 13 Lys residues within NS5A (genotype 2a, JFH1 strain) were conserved compared to those of HCV (genotype 1b, Con1 strain). The cell-based ISGylation assay revealed that the K26 residue in the amphipathic helix (AH) domain and the K139 residue in domain I of NS5A (2a, JFH1) had the potential to accept ISGylation. Use of the HCV replicon carrying luciferase gene revealed that the K26 residue but not K139 residue of NS5A (2a, JFH1) was important for HCV RNA replication. Furthermore, cell culture HCV revealed that the mutation with the K26 residue in combination with K139 or K166 on NS5A (2a, JFH1) resulted in complete abolishment of viral propagation, suggesting that the K26 residue collaborates with either the K139 residue or K166 residue for efficient HCV propagation. Taken together, these results suggest that HCV NS5A protein has the potential to accept ISGylation via specific Lys residues, involving efficient viral propagation in a genotype-specific manner.

Vitamin K-Dependent γ-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

Immunohistochemical Mapping of Bcl9 Using Two Antibodies that Recognize Different Epitopes Is Useful to Characterize Juvenile Development of Hepatocellular Carcinoma in Myanmar.

B-cell lymphoma 9 (Bcl9) is the core component of Wnt/ß-catenin signaling and overexpressed in nuclei of various tumors, including hepatocellular carcinoma (HCC). However, the extent of Bcl9 expression relative to HCC differentiation stage and its functional aspects are poorly understood. In this study, we examined the expression pattern of Bcl9 immunohistochemically, using two anti-Bcl9 antibodies; one was a conventional polyclonal-antibody (anti-Bcl9ABC) against amino acid no.800-900 of human-Bcl9, while the other (anti-Bcl9BIO) was against amino acid no.50-200, covering Pygopus-binding sites of Bcl9. Immunohistochemistry using anti-Bcl9BIO demonstrated distinctive staining in the cytoplasm, while the anti-Bcl9ABC signal was detected in both cytoplasm and nuclei of HCC cells, reflecting different states of Bcl9 function because Pygopus-binding to Bcl9 is essential to exert its function together with ß-catenin in nucleus. Quantitative analysis revealed a significantly higher immunohistochemical-score by anti-Bcl9BIO in normal liver comparing various differentiation grades of HCC (P < 0.004), whereas no significant difference was noted with anti-Bcl9ABC. Interestingly, immunohistochemical-score of anti-Bcl9BIO in patients aged < 40 years was significantly lower than that of ≥ 40 years group (P < 0.01). The results indicated that anti-Bcl9BIO detected cytoplasmic Bcl9, which does not bind to Pygopus suggesting it could be a useful indicator for development of HCC in young Myanmar patients.

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.

Enhanced osseointegration by the chemotactic activity of plasma fibronectin for cellular fibronectin positive cells.

Plasma fibronectin (pFN) is known to regulate cell growth, differentiation or survival of osteoblasts in vitro. It is also speculated to be important for the early phase of osseointegration, however, its actual in vivo behavior is unknown. The objective of this study is to clarify the role of pFN during osseointegration. We developed a titanium ion-plated acrylic implant (Ti-acryl) for thin sectioning without removal of the implant. Either Ti-acryl or pFN-coated Ti-acryl (FN-Ti-acryl) was implanted in the mouse femur. Samples were taken on days 1-7 and on day 14 after the operation, and were decalcified and paraffin embedded. The bone healing process and immunofluorescence localization of pFN and cellular fibronectin (cFN), a marker for fibroblastic cells were examined. Simultaneously, the effect of pFN on chemotaxis, proliferation and differentiation of bone marrow stromal cells (BMSCs) was analyzed in vitro. The in vivo results showed that faster direct bone formation was seen for the FN-Ti-acryl group compared to the Ti-acryl group. The in vitro results showed that pFN significantly promoted BMSCs chemotaxis, however, had no effect on proliferation or differentiation. The results indicate that pFN regulated chemotaxis of osteogenic cells and coating the implant with pFN enhanced earlier osseointegration.