Nanometer-thin TiO2 enhances skeletal muscle cell phenotype and behavior

Ken Ishizaki*, Yoshihiko Sugita*, Fuminori Iwasa, Hajime Minamikawa, Takeshi Ueno, Masahiro Yamada, Takeo Suzuki, Takahiro OgawaLaboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Los Angeles, CA, USA*Authors contributed equally to this workBackground: The independent role of the surface chemistry of titanium in determining its biological properties is yet to be determined. Although titanium implants are often in contact with muscle tissue, the interaction of muscle cells with titanium is largely unknown. This study tested the hypotheses that the surface chemistry of clinically established microroughened titanium surfaces could be controllably varied by coating with a minimally thin layer of TiO2 (ideally pico-to-nanometer in thickness) without altering the existing topographical and roughness features, and that the change in superficial chemistry of titanium is effective in improving the biological properties of titanium.Methods and results: Acid-etched microroughened titanium surfaces were coated with TiO2 using slow-rate sputter deposition of molten TiO2 nanoparticles. A TiO2 coating of 300 pm to 6.3 nm increased the surface oxygen on the titanium substrates in a controllable manner, but did not alter the existing microscale architecture and roughness of the substrates. Cells derived from rat skeletal muscles showed increased attachment, spread, adhesion strength, proliferation, gene expression, and collagen production at the initial and early stage of culture on 6.3 nm thick TiO2-coated microroughened titanium surfaces compared with uncoated titanium surfaces.Conclusion: Using an exemplary slow-rate sputter deposition technique of molten TiO2 nanoparticles, this study demonstrated that titanium substrates, even with microscale roughness, can be sufficiently chemically modified to enhance their biological properties without altering the existing microscale morphology. The controllable and exclusive chemical modification technique presented in this study may open a new avenue for surface modifications of titanium-based biomaterials for better cell and tissue affinity and reaction.Keywords: nanotechnology, orthopedic implants, molten TiO2 nanoparticles, surface chemistr

Breakthroughs in Implant Dentistry

The osseointegration capacity of implant surfaces deteriorates over time. However, the treatment of titanium surfaces with ultraviolet (UV) light restores the original properties of the surface and causes considerable acceleration in the process of osseointegration. This study reviews two recent findings: the aging-like time-dependent biological degradation of titanium surfaces and the discovery of UV photofunctionalization as a solution to this phenomenon. This technology and the associated knowledge herald a new age of implant treatment and provide a novel concept of osseointegration in the science and therapeutics of implant dentistry. In addition, we expect to revolutionize clinical implant therapy through these new concepts

Temperature diagnostics for the winter mesopause region estimated from N2+ (0,0) Meinel band at 1.1 µm observed by the NIRAS-2 and the NIRAC

The 14th Symposium on Polar Science/Interdisciplinary sessions [IW] Whole Atmosphere, Thu. 16 Nov. / 3F Multipurpose Conference room, Institute of Statistical Mathematic

Downregulation of Carbonic Anhydrase IX Promotes <em>Col10a1</em> Expression in Chondrocytes

<div><p>Carbonic anhydrase (CA) IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO₂. While it is known that CA IX is distributed in human embryonic chondrocytes, its role in chondrocyte differentiation has not been reported. In the present study, we found that <i>Car9</i> mRNA and CA IX were expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation <i>in vitro</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#s1" target="_blank">Introduction</a> of <i>Car9</i> siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of <i>Col10a1</i>, the gene for type X collagen α-1 chain, and <i>Epas1</i>, the gene for hypoxia-responsible factor-2α (HIF-2α), both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of <i>Col10a1</i> and <i>Epas1</i>, while the transcription of <i>Col2a1</i> and <i>Acan</i> were up-regulated. Although HIF-2α has been reported to be a potent activator of <i>Col10a1</i> transcription, <i>Epas1</i> siRNA did not suppress <i>Car9</i> siRNA-induced increment in <i>Col10a1</i> expression, indicating that down-regulation of CA IX induces the expression of <i>Col10a1</i> in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by <i>Car9</i> siRNA. Furthermore, the expression of <i>Col10a1</i> mRNA after <i>Car9</i> silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of <i>Col10a1</i> expression by down-regulation of <i>Car9</i>, more detailed study is required to clarify the role of CA IX in regulation of <i>Col10a1</i> expression in chondrocytes.</p> </div

Intracellular signaling cascades involved in regulation of <i>Col10a1</i> expressions by CA IX.

<p><i>Car9</i> siRNA or control siRNA was introduced into primary chondrocytes. <b>A.</b> At 24 hours after introduction of the siRNAs, the culture medium was changed to that containing the JNK inhibitor SP600125 (30 µM), p38 MAPK (p38) inhibitor SB202190 (20 µM), PKA inhibitor H89 (10 µM), PDE inhibitor IBMX (0.1 mM), PI3K inhibitor LY294002 (20 µM), Src kinase Inhibitor I (20 µM), or hedgehog (Hh) inhibitor Jervine (10 µM). After additional incubation for 24 hours, the mRNA expressions of <i>Epas1</i>, <i>Col10a1</i>, and <i>Gapdh</i> were examined by RT-PCR. <b>B.</b> At 36 hours after introduction of <i>Car9</i> or control siRNA to primary chondrocytes, cAMP was extracted with 0.1 M HCl and its level was determined using an EIA Kit, then corrected for cellular protein. Data are expressed as the mean ± SD of 6 experiments. <b>C</b>. At 36 hours after introduction of <i>Car9</i> or control siRNA to primary chondrocytes, PKA activity was determined and corrected for cellular protein. Data are expressed as the mean ± SD of 13 determinations. <b>D-F</b>. At 24 hours after introduction of <i>Car9</i> or control siRNA, chondrocytes were treated for 24 hours with 0.2 mM Br-cAMP. The expression levels of mRNAs for <i>Car9</i> (<b>D</b>), <i>Col10a1</i> (<b>E</b>), and <i>Epas1</i> (<b>F</b>) were analyzed by real-time RT-PCR. Data are expressed as the mean ± SD of 4 experiments. <i>P</i>-values determined by two-tailed Mann-Whitney <i>U</i>-test are indicated.</p

Effects of forced expression of CA IX on chondrocyte proliferation and mRNA expression of <i>Col2a1</i>, <i>Acan</i>, <i>Col10a1</i>, and <i>Epas1</i>.

<p>Primary mouse chondrocytes were transfected with a CA IX-expression plasmid (+) or its control plasmid (-). A. At 48 hours after transfection, the expression of CA IX (58kDa) was assessed by western blot analysis using anti-mouse CA IX antibody. CA IX band is indicated by an arrowhead. B. Proliferation of chondrocytes introduced with CA IX-expression (filled square) and control (unfilled circle) plasmids was assessed spectrophotometrically using CellTiter 96^® Aqueous One Solution. Data are expressed as the mean ± SD (n = 4). At each time point, Mann-Whitney <i>U</i>-test with Bonferroni correction was performed to evaluate the difference between control plasmid- and CAIX-expression plasmid-transfected cells. No significant difference between the values was indicated at any time point (α = 0.01). C-F. The expressions of <i>Col2a1</i> (C), <i>Acan</i> (D), <i>Col10a1</i> (E), and <i>Epas1</i> (F) were quantitatively analyzed by real-time RT-PCR, with the expression level of each normalized to that of <i>Gapdh</i>. Data are expressed by boxplots (n = 6) for fold changes caused by introduction of <i>Car9</i> siRNA (the sample maximum, the upper quartile, the median, the lower quartile, and the minimum observation). <i>P</i>-values determined by two-tailed Mann-Whitney <i>U</i>-test are indicated.</p