Mechanical, bioactive, and long-lasting antibacterial properties of a Ti scaffold with gradient pores releasing iodine ions.

The ideal bone implant would effectively prevent aseptic as well as septic loosening by minimizing stress shielding, maximizing bone ingrowth, and preventing implant-associated infections. Here, a novel gradient-pore-size titanium scaffold was designed and manufactured to address these requirements. The scaffold features a larger pore size (900 µm) on the top surface, gradually decreasing to small sizes (600 µm to 300 µm) towards the center, creating a gradient structure. To enhance its functionality, the additively manufactured scaffolds were biofunctionalized using simple chemical and heat treatments so as to incorporate calcium and iodine ions throughout the surface. This unique combination of varying pore sizes with a biofunctional surface provides highly desirable mechanical properties, bioactivity, and notably, long-lasting antibacterial activity. The target mechanical aspects, including low elastic modulus, high compression, compression-shear, and fatigue strength, were effectively achieved. Furthermore, the biofunctional surface exhibits remarkable in vitro bioactivity and potent antibacterial activity, even under conditions specifically altered to be favorable for bacterial growth. More importantly, the integration of small pores alongside larger ones ensures a sustained high release of iodine, resulting in antimicrobial activity that persisted for over three months, with full eradication of the bacteria. Taken together, this gradient structure exhibits obvious superiority in combining most of the desired properties, making it an ideal candidate for orthopedic and dental implant applications.

Real-time scanning electron microscopy of unfixed tissue in the solution using a deformable and electron-transmissive film.

It is difficult to use scanning electron microscopy to observe the structure and movement of biological tissue immersed in the solution. To enable such observations, we created a highly deformable and electron-transmissive polyimide film that can withstand the pressure difference between the high-vacuum electron column and the atmospheric-pressure sample chamber. With this film, we used scanning electron microscopy to measure the intrinsic fine structure and movement of the contractile fibers of excised mouse heart immersed in physiological solutions. Our measurements revealed that the excised heart is a dynamic tissue that undergoes relaxation oscillation based on a three-dimensional force balance.

Iodine-Loaded Calcium Titanate for Bone Repair with Sustainable Antibacterial Activity Prepared by Solution and Heat Treatment.

In the orthopedic and dental fields, simultaneously conferring titanium (Ti) and its alloy implants with antibacterial and bone-bonding capabilities is an outstanding challenge. In the present study, we developed a novel combined solution and heat treatment that controllably incorporates 0.7% to 10.5% of iodine into Ti and its alloys by ion exchange with calcium ions in a bioactive calcium titanate. The treated metals formed iodine-containing calcium-deficient calcium titanate with abundant Ti-OH groups on their surfaces. High-resolution XPS analysis revealed that the incorporated iodine ions were mainly positively charged. The surface treatment also induced a shift in the isoelectric point toward a higher pH, which indicated a prevalence of basic surface functionalities. The Ti loaded with 8.6% iodine slowly released 5.6 ppm of iodine over 90 days and exhibited strong antibacterial activity (reduction rate >99%) against methicillin-resistant Staphylococcus aureus (MRSA), S. aureus, Escherichia coli, and S. epidermidis. A long-term stability test of the antibacterial activity on MRSA showed that the treated Ti maintained a >99% reduction until 3 months, and then it gradually decreased after 6 months (to a 97.3% reduction). There was no cytotoxicity in MC3T3-E1 or L929 cells, whereas apatite formed on the treated metal in a simulated body fluid within 3 days. It is expected that the iodine-carrying Ti and its alloys will be particularly useful for orthopedic and dental implants since they reliably bond to bone and prevent infection owing to their apatite formation, cytocompatibility, and sustainable antibacterial activity.

Bioactivation Treatment with Mixed Acid and Heat on Titanium Implants Fabricated by Selective Laser Melting Enhances Preosteoblast Cell Differentiation.

Selective laser melting (SLM) is a promising technology capable of producing individual characteristics with a high degree of surface roughness for implants. These surfaces can be modified so as to increase their osseointegration, bone generation and biocompatibility, features which are critical to their clinical success. In this study, we evaluated the effects on preosteoblast proliferation and differentiation of titanium metal (Ti) with a high degree of roughness (Ra = 5.4266 ± 1.282 µm) prepared by SLM (SLM-Ti) that was also subjected to surface bioactive treatment by mixed acid and heat (MAH). The results showed that the MAH treatment further increased the surface roughness, wettability and apatite formation capacity of SLM-Ti, features which are useful for cell attachment and bone bonding. Quantitative measurement of osteogenic-related gene expression by RT-PCR indicated that the MC3T3-E1 cells on the SLM-Ti MAH surface presented a stronger tendency towards osteogenic differentiation at the genetic level through significantly increased expression of Alp, Ocn, Runx2 and Opn. We conclude that bio-activated SLM-Ti enhanced preosteoblast differentiation. These findings suggest that the mixed acid and heat treatment on SLM-Ti is promising method for preparing the next generation of orthopedic and dental implants because of its apatite formation and cell differentiation capability.

Impact of Surface Potential on Apatite Formation in Ti Alloys Subjected to Acid and Heat Treatments.

Titanium metal (Ti) and its alloys are widely used in orthopedic and dental fields. We have previously shown that acid and heat treatment was effective to introduce bone bonding, osteoconduction and osteoinduction on pure Ti. In the present study, acid and heat treatment with or without initial NaOH treatment was performed on typical Ti-based alloys used in orthopedic and dental fields. Dynamic movements of alloying elements were developed, which depended on the kind of treatment and type of alloy. It was found that the simple acid and heat treatment enriched/remained the alloying elements on Ti-6Al-4V, Ti-15Mo-5Zr-3Al and Ti-15Zr-4Nb-4Ta, resulting in neutral surface charges. Thus, the treated alloys did not form apatite in a simulated body fluid (SBF) within 3 days. In contrast, when the alloys were subjected to a NaOH treatment prior to an acid and heat treatment, alloying elements were selectively removed from the alloy surfaces. As a result, the treated alloys became positively charged, and formed apatite in SBF within 3 days. Thus, the treated alloys would be useful in orthopedic and dental fields since they form apatite even in a living body and bond to bone.

Fabrication of dense α-alumina layer on Ti-6Al-4V alloy hybrid for bearing surfaces of artificial hip joint.

Recent advances in hip replacements are focused towards producing reliable bearing surfaces to enhance their longevity. In this perspective, progressive attempts have been made to improve the wear resistance of polyethylene to eliminate osteolysis and mechanical reliability of brittle alumina ceramics, but in vain. It is proposed that both high wear resistance and mechanical reliability can be retained if a thin layer of dense alumina is formed onto high toughness Ti-6Al-4V alloy. For this purpose, we devised a unique methodology in which a layer of Al metal was deposited onto the Ti alloy substrate by cold spraying (CS), followed by a heat treatment to form Al3Ti reaction layer at their interface to improve adhesion and subsequent micro-arc oxidation (MAO) treatment to transform Al to alumina layer. An optimal MAO treatment of cold sprayed Al formed an adherent and dense α-alumina layer with high Vickers hardness matching with that of sintered alumina used as a femoral head. Structure-phase-property relationships in dense α-alumina layer have been revealed and discussed in the light of our research findings. The designed alumina/Ti alloy hybrid might be a potential candidate for reliable bearing surfaces of artificial hip joint.

Novel artificial hip joint: A layer of alumina on Ti-6Al-4V alloy formed by micro-arc oxidation.

In many hip replacement surgeries, monolithic alumina is used as a femoral head due to its high wear resistance. However, it is liable to fracture under load bearing operations in artificial joints. We propose a promising way to overcome this limitation by forming a dense alumina layer onto a relatively tough substrate such as Ti-6Al-4V alloy to obtain high wear resistance on a material that can sustain relatively high toughness. For this purpose, Al metal powders were deposited onto Ti-6Al-4V alloy by cold spraying in N2 atmosphere. Interfacial adhesion between Al and the Ti alloy was improved by the formation of a reaction layer of Al3Ti between them by heating at 640 °C for 1h in air. Subsequently, micro-arc oxidation treatment was performed to oxidize Al. The oxidized layer was composed of an outer porous layer of γ-alumina and inner-most dense layer of α-alumina. The α-alumina layer was almost fully densified and exhibited high Vickers hardness almost equal to that of alumina ceramics used as the femoral head. Thus, the newly developed dense alumina/Ti alloy can be potentially used to produce the reliable bearing surfaces of artificial hip joint.

Effect of Ca contamination on apatite formation in a Ti metal subjected to NaOH and heat treatments.

It has long been known that titanium (Ti) metal bonds to living bone through an apatite layer formed on its surface in the living body after it had previously been subjected to NaOH and heat treatments and as a result had formed sodium titanate on its surface. These treatments were applied to a porous Ti metal layer on a total hip joint and the resultant joint has been in clinical use since 2007. It has been also demonstrated that the apatite formation on the treated Ti metal in the living body also occurred in an acelullar simulated body fluid (SBF) with ion concentrations nearly equal to those of the human blood plasma, and hence bone-bonding ability of the treated Ti metal can be evaluated using SBF in vitro. However, it was recently found that certain Ti metals subjected to the same NaOH and heat treatments display apatite formation in SBF which is decreased with the increasing volume of the NaOH solution used in some cases. This indicates that bone-bonding ability of the treated Ti metal varies with the volume of the NaOH solution used. In the present study, this phenomenon was systematically investigated using commercial NaOH reagents and is considered in terms of the structure and composition of the surface layers of the treated Ti metals. It was found that a larger amount of the calcium contamination in the NaOH reagent is concentrated on the surface of the Ti metal during the NaOH treatment with an increasing volume of the NaOH solution, and that this inhibited apatite formation on the Ti metal in SBF by suppressing Na ion release from the sodium titanate into the surrounding fluid. Even a Ca contamination level of 0.0005 % of the NaOH reagent was sufficient to inhibit apatite formation. On the other hand, another NaOH reagent with a nominal purity of just 97 % did not exhibit any such inhibition, since it contained almost no Ca contamination. This indicates that NaOH reagent must be carefully selected for obtaining reliable bone-bonding implants of Ti metal by the NaOH and heat treatments.

Formation of a bioactive calcium titanate layer on gum metal by chemical treatment.

The so-called gum metal with the composition Ti-36Nb-2Ta-3Zr-0.3O is free from cytotoxic elements and exhibits a low elastic modulus as well as high mechanical strength. In the present study, it was shown that this alloy exhibited a high capacity for apatite formation in a simulated body fluid when subjected to 1 M NaOH treatment, 100 mM CaCl(2) treatment, heat treatment at 700°C, and then hot water treatment. The high apatite formation was attributed to the CaTi(2)O(5) which was precipitated on its surface, and found to be maintained even in a humid environment over a long period. The treated surface exhibited high scratch resistance, which is likely to be useful in clinical applications. The surface treatment had little effect on the unique mechanical properties described above. These results show that gum metal subjected to the present surface treatments exhibits a high potential for bone-bonding, which will be useful in orthopedic and dental implants.

Preparation of bioactive Ti-15Zr-4Nb-4Ta alloy from HCl and heat treatments after an NaOH treatment.

Ti-15Zr-4Nb-4Ta alloy does not contain any cytotoxic elements and has a high mechanical strength. Water or HCl and heat treatments were applied to this alloy after NaOH treatment to form a bioactive titanium oxide layer with a nanometer scale roughness on its surface. The nanometer scale roughness was formed on the surface after the first NaOH treatment and remained, even after a subsequent water or HCl and heat treatment. A layer that was mainly composed of anatase was formed on the surface after the heat treatment. Thus, the treated alloy showed a high apatite-forming ability in an SBF, as well as a high scratch resistance. Its high apatite-forming ability was attributed to its positive surface charge. The same alloy subjected to a heat treatment without a water or HCl treatment after the NaOH treatment did not show an apatite-forming ability. This was attributed to a too slow release rate of sodium ions from the surface in an SBF. Ti-15Zr-4Nb-4Ta alloy samples subjected to a water or HCl and heat treatment after the NaOH treatment are expected to be useful as orthopedic and dental implants, since they can form an apatite layer on their surface in a living body and bond to living bone through this apatite layer.

Prediction of osteoconductive activity of modified potassium fluorrichterite glass-ceramics by immersion in simulated body fluid.

Potassium fluorrichterite (KNaCaMg(5)Si(8)O(22)F(2)) glass-ceramics were modified by either increasing the concentration of calcium (GC5) or by the addition of P(2)O(5) (GP2). The stoichiometric composition (GST), GC5 and GP2 were soaked in simulated body fluid (SBF) along with 45S5-type bioglass as a control. After immersion, surface analyses were performed using thin-film X-ray diffraction (TF-XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and Fourier-transform infrared (reflection) spectroscopy (FT-IR). All compositions showed the formation of a calcium phosphate rich surface layer in SBF; GST, GP2 and the bioglass control within 7 days of immersion and GC5 after 14 days. It was concluded that all compositions were likely to be osteoconductive in vivo, with GP2 providing the best performance in terms of the combination of rapid formation of the surface layer and superior mechanical properties. This glass-ceramic system has potential as a load bearing bioceramic for fabrication of medical devices intended for skeletal tissue repair.

Positively charged bioactive Ti metal prepared by simple chemical and heat treatments.

A highly bioactive bone-bonding Ti metal was obtained when Ti metal was simply heat-treated after a common acid treatment. This bone-bonding property was ascribed to the formation of apatite on the Ti metal in a body environment. The formation of apatite on the Ti metal was induced neither by its surface roughness nor by the rutile phase precipitated on its surface, but by its positively charged surface. The surface of the Ti metal was positively charged because acid groups were adsorbed on titanium hydride formed on the Ti metal by the acid treatment, and remained even after the titanium hydride was transformed into titanium oxide by the subsequent heat treatment. These results provide a new principle based on a positively charged surface for obtaining bioactive materials.

Preparation of bioactive Ti metal surface enriched with calcium ions by chemical treatment.

A calcium solution treatment was applied to a NaOH-treated titanium metal to give it bioactivity, scratch resistance and moisture resistance. The titanium metal was soaked in a 5 M NaOH solution and then a 100 mM CaCl(2) solution to incorporate Ca(2+) ions into the titanium metal surface by ion exchange. This treated titanium metal was subsequently heated at 600 degrees C and soaked in hot water at 80 degrees C. The NaOH treatment incorporated approximately 5 at.% Na(+) ions into the Ti metal surface. These Na(+) ions were completely replaced by Ca(2+) ions by the CaCl(2) treatment. The number of Ca(2+) ions remained even after subsequent heat and water treatments. Although the NaOH-CaCl(2)-treated titanium metal showed slightly higher apatite-forming ability in a simulated body fluid than the NaOH-treated titanium metal, it lost its apatite-forming ability during the heat treatment. However, subsequent water or autoclave treatment restored the apatite-forming ability of the NaOH-CaCl(2)-heat-treated titanium metal. Although the apatite-forming ability of the NaOH-heat-treated titanium metal decreased dramatically when it was kept at high humidity, that of NaOH-CaCl(2)-heat-water-treated titanium metal was maintained even in the humid environment. The heat treatment increased the critical scratch resistance of the surface layer of the NaOH-CaCl(2)-treated titanium metal remarkably, and it did not deteriorate on subsequent water treatment.

Apatite-forming ability of Ti-15Zr-4Nb-4Ta alloy induced by calcium solution treatment.

Ti-15Zr-4Nb-4Ta alloy free from cytotoxic elements shows high mechanical strength and high corrosion resistance. However, simple NaOH and heat treatments cannot induce its ability to form apatite in the body environment. In the present study, this alloy was found to exhibit high apatite-forming ability when it was treated with NaOH and CaCl(2) solutions, and then subjected to heat and hot water treatments to form calcium titanate, rutile, and anatase on its surface. Its high apatite-forming ability was maintained even in 95% relative humidity at 80 degrees C after 1 week. The surface layer of the treated alloy had scratch resistance high enough for handling hard surgical devices. Thus, the treated alloy is believed to be useful for orthopedic and dental implants.

Effect of HCl concentrations on apatite-forming ability of NaOH-HCl- and heat-treated titanium metal.

Titanium (Ti) metal was treated with water or HCl solutions after 5 M NaOH solution treatment and then subjected to heat treatment at 600 degrees C. The apatite-forming abilities of the treated Ti metals were examined in simulated body fluid. The apatite-forming ability of the Ti metal subjected to NaOH, water and heat treatment was lower than that of just NaOH and heat treatments. Ti metals subjected to NaOH, HCl and heat treatment showed apatite-forming abilities, which increased with increasing HCl concentrations up to the same level as that of NaOH- and heat-treated Ti metal. The former did not show a decrease in its apatite-forming ability, even in a humid environment for a long period, whereas the latter decreased its ability. The increase in the apatite-forming ability with increasing HCl concentrations suggests a different mechanism of apatite formation from that previously proposed.

Mechanical, setting, and biological properties of bone cements containing micron-sized titania particles.

In this study, polymethylmethacrylate-based composite cements containing 40-55.6 wt% micron-sized titania (titanium oxide) particles were developed, and their mechanical, setting, and biological properties evaluated. Three types of composite cement containing 40, 50, and 55.6 wt% silanized titania were designated ST2-40c, ST2-50c, and ST2-56c, respectively. In animal experiments, ST2-50c and ST2-56c were implanted into rat tibiae and solidified in situ. An affinity index was used to evaluate osteoconductivity. Compressive and bending strength of ST2-56c was 147.7+/-3.2 and 69.3+/-7.4; those of the other cements exceeded 100 MPa and 50 MPa, respectively. The affinity indices of ST2-56c were 42.1+/-12.9 at six weeks and 53.4+/-16.6 at 12 weeks, and were significantly higher than for ST2-50c and a commercial PMMA bone cement within 12 weeks. Our data indicate that bone cement containing micron-sized titania particles can be applied to prosthesis fixation as well as vertebroplasty, and ST2-56c is a good candidate cement.

Mechanical properties and apatite forming ability of TiO2 nanoparticles/high density polyethylene composite: Effect of filler content.

Composite materials consisting of TiO(2) nanoparticles and high-density polyethylene (HDPE), designated hereafter as TiO(2)/HDPE, were prepared by a kneading and forming process. The effect of TiO(2) content on the mechanical properties and apatite forming ability of these materials was studied. Increased TiO(2) content resulted in an increase in bending strength, yield strength, Young's modulus and compressive strength (bending strength = 68 MPa, yield strength = 54 MPa, Young's modulus = 7 GPa, and compressive strength = 82 MPa) at 50 vol% TiO(2). The composite with 50 vol% TiO(2) shows a similar strength and Young's modulus to human cortical bone. The TiO(2)/HDPE composites with different TiO(2) contents were soaked at 36.5 degrees C for up to 14 days in a simulated body fluid (SBF) whose ion concentrations were nearly equal to those of human blood plasma. The apatite forming ability, which is indicative of bioactivity, increased with TiO(2) content. Little apatite formation was observed for the TiO(2)/HDPE composite with 20 vol% content. However, in the case of 40 vol% TiO(2) content and higher, the apatite layers were formed on the surface of the composites within 7 days. The most potent TiO(2) content for a bone-repairing material was 50 vol%, judging from the mechanical and biological results. This kind of bioactive material with similar mechanical properties to human cortical bone is expected to be useful as a load bearing bone substitute in areas such as the vertebra and cranium.

Effects of photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on physical properties of cross-linked polyethylene in artificial hip joints.

Osteolysis caused by wear particles from polyethylene in the artificial hip joints is a serious issue. We have used photo-induced radical graft polymerization to graft 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of cross-linked polyethylene (CLPE-g-MPC) in order to reduce friction and wear at the bearing surface of the joint. The physical and mechanical properties of CLPE and CLPE-g-MPC were not significantly different, expect that the friction coefficient of untreated CLPE cups was 0.0075, compared with 0.0009 for CLPE-g-MPC cup, an 88% reduction. After 3.0 x 10(6) cycles in the hip joint simulator test, we could not observe any wear of CLPE-g-MPC cups. We concluded that the advantage of photo-induced radical graft polymerization technique was that the grafted MPC polymer gave a high lubricity only on the surface and has no effect on the bulk properties of the CLPE substrate.

Enhanced wear resistance of modified cross-linked polyethylene by grafting with poly(2-methacryloyloxyethyl phosphorylcholine).

We developed a cross-linked polyethylene (CLPE) modified with a phospholipid polymer in order to address the serious problem of osteolysis caused by wear particles derived from the polyethylene components of artificial hip joints. Our goal of preventing aseptic loosening could be achieved by avoiding any formation of CLPE wear particles or suppressing the activation of cell systems by the wear particles. We investigated the surface and wear resistance properties of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer grafted onto the surface of CLPE (CLPE-g-MPC). The relative density of MPC polymer chains was determined by the P-O group index. Generally, polymerization times correspond to the number of polymer chains in radical polymerization. After 3.0 x 10(6) cycles in a hip joint simulator test, the steady wear rates of the untreated CLPE and CLPE-g-MPC cups with a low P-O group index were as high as 4 mg/10(6) cycles; those of the CLPE-g-MPC cups with high P-O group indexes, that is, 0.46 and 0.48, markedly decreased to -1.12 and 0.16 mg/10(6) cycles, respectively. Therefore, the grafting of an MPC polymer with high density would be essential in order to maintain the long-term wear resistance of CLPE-g-MPC as an orthopedic bearing material.

How useful is SBF in predicting in vivo bone bioactivity?

The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. However, the validity of this method for evaluating bone-bonding ability has not been assessed systematically. Here, the history of SBF, correlation of the ability of apatite to form on various materials in SBF with their in vivo bone bioactivities, and some examples of the development of novel bioactive materials based on apatite formation in SBF are reviewed. It was concluded that examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material, and the number of animals used in and the duration of animal experiments can be reduced remarkably by using this method.