Gamma ray treatment enhances bioactivity and osseointegration capability of titanium.

The time-dependent degradation of titanium bioactivity (i.e., the biological aging of titanium) has been reported in previous studies. This phenomenon is caused by the loss of hydrophilicity and the inevitable occurrence of progressive contamination of titanium surfaces by hydrocarbons. In this study, we tested the hypothesis that gamma ray treatment, owing to its high energy to decompose and remove organic contaminants, enhances the bioactivity and osteoconductivity of titanium. Titanium disks were acid-etched and stored for 4 weeks. Rat bone marrow-derived osteoblasts (BMOs) were cultured on titanium disks with or without gamma ray treatment (30 kGy) immediately before experiments. The cell density at day 2 increased by 50% on gamma-treated surfaces, which reflected the 25% higher rate of cell proliferation. Osteoblasts on gamma-treated surfaces showed 30% higher alkaline phosphatase activity at day 5 and 60% higher calcium deposition at day 20. The strength of in vivo bone-implant integration increased by 40% at the early healing stage of week 2 for gamma-treated implants. Gamma ray-treated surfaces regained hydrophilicity and showed a lower percentage of carbon (35%) as opposed to 48% on untreated aged surfaces. The data indicated that gamma ray pretreatment of titanium substantially enhances its bioactivity and osteoconductivity, in association with the significant reduction in surface carbon and the recovery of hydrophilicity. The results suggest that gamma ray treatment could be an effective surface enhancement technology to overcome biological aging of titanium and improve the biological properties of titanium implants.

Alleviation of commercial collagen sponge- and membrane-induced apoptosis and dysfunction in cultured osteoblasts by an amino acid derivative.

PURPOSE: The objectives of this in vitro study were to determine whether the commercial collagen material used in bone augmentation procedures induces oxidative stress-mediated adverse effects on the viability and function of osteoblasts and to determine whether N-acetyl cysteine (NAC), an antioxidant amino acid derivative, can alleviate these effects. MATERIALS AND METHODS: Commercial collagen sponge (Collaplug) and membrane (BioGide) were treated with NAC. Rat calvaria-derived osteoblasts were directly seeded on these materials with or without NAC pretreatment. Cytotoxic evaluation was performed by flowcytometric cell viability assay, confocal laser microscopic analysis of attached cell morphology and reactive oxygen species (ROS) localization, and alkaline phosphatase staining. RESULTS: Cell viability was less than 40% on both collagen sponge and membrane 24 hours after seeding and increased to 50% with NAC pretreatment. Cell death was characterized by apoptosis. Colonization of attached cells was sparse on the untreated sponge and membrane on day 1, and the cells were round, small, and filled with intense and closely packed intracellular ROS. In contrast, NAC-pretreated material had dense cell colonies consisting of well-spread osteoblasts and fully developing cytoskeleton and cellular processes with little ROS generation. On day 7 of culture, NAC-pretreated collagen sponge and membrane yielded an expanded alkaline phosphatase-positive area occupying 60% and 80% of the surface area, respectively, whereas the untreated collagen materials had limited alkaline phosphatase activity (7% or less). CONCLUSIONS: Commercial collagen sponge and membrane induced considerable cell death, impaired initial function, and generated extraordinary intracellular ROS in attached osteoblasts, whereas NAC pretreatment substantially ameliorated these effects. The potential benefits of NAC's detoxifying capacity on bone regeneration using collagen matrix materials in an animal model should be confirmed with further study.

Effect of ultraviolet photoactivation of titanium on osseointegration in a rat model.

PURPOSE: The objective of this study was to determine whether ultraviolet (UV) light treatment of titanium implants could enhance osseointegration to sufficiently overcome the negative aspects of shorter implants in a rat femur model. MATERIALS AND METHODS: Acid-etched miniature titanium implants with lengths of 2 mm (longer implants) and 1.2 mm (shorter implants) were prepared. Some of these implants were treated with UV light for 48 hours prior to surgery. The strength of osseointegration generated by these implants was evaluated using a biomechanical implant push-in test in a rat model. Peri-implant osteogenesis was examined by scanning electron microscopy for tissue morphology and energy dispersive x-ray spectroscopy for elemental composition. RESULTS: Push-in test values for the longer implants were 80% and 100% greater than those of the shorter implants at weeks 4 and 8 of healing, respectively. UV treatment of the shorter implants significantly increased their push-in value, resulting in a 100% higher value than untreated longer implants at week 2 and a push-in value that was equivalent to that of the untreated longer implants at weeks 4 and 8. Scanning electron micrographs and energy dispersive x-ray spectroscopic examinations after push-in testing revealed that the UV-treated implant surfaces were covered more extensively by bone or tissue remnants containing calcium and phosphorous than the untreated surfaces. The titanium surfaces were converted from hydrophobic to superhydrophilic status after UV treatment, although the cause-result relationship between the acquired superhydrophilicity and biologic effects remained unclear. CONCLUSIONS: Within the limits of this investigation, UV light pretreatment substantially enhanced the osseointegration capacity of acid-etched titanium implants. The deficiencies of osseointegration in implants with a 40% shorter length were overcome by UV treatment in the rat model using miniature implants. These results need to be confirmed in other animal models and implants that more closely resemble human dental implants to determine the true clinical significance.

Nonvolatile buffer coating of titanium to prevent its biological aging and for drug delivery.

The osseointegration capability of titanium decreases over time. This phenomenon, defined as biological aging of titanium, is associated with the disappearance of hydrophilicity and the progressive accumulation of hydrocarbons on titanium surfaces. The objective of this study was to examine whether coating of titanium surfaces with 4-(2-Hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, a nonvolatile zwitterionic chemical buffering agent, could prevent the time-dependent degradation of the bioactivity of titanium. Commercially pure titanium samples, prepared as disks and cylinders, were acid-etched with H(2)SO(4). A third of the samples were used for experiments immediately after processing (new surfaces), while another third were stored under dark ambient conditions for 3 months (3-month-old surfaces). The remaining third were coated with HEPES after acid-etching and were stored for 3 months (HEPES-coated 3-month-old surfaces). The 3-month-old surfaces were hydrophobic, while new and HEPES-coated 3-month-old surfaces were superhydrophilic. Protein adsorption and the number of osteoblasts attached during an initial culture period were substantially lower for 3-month-old surfaces than for new and HEPES-coated 3-month-old surfaces. Alkaline phosphatase activity and calcium deposition in osteoblast cultures were reduced by more than 50% on 3-month-old surfaces compared to new surfaces, whereas such degradation was not found on HEPES-coated 3-month-old surfaces. The strength of in vivo bone-implant integration for 3-month-old implants, evaluated by the push-in test, was 60% lower than that for new implants. The push-in value of HEPES-coated 3-month-old implants was equivalent to that of new implants. Coating titanium surfaces with HEPES containing an antioxidant amino acid derivative, N-acetyl cysteine (NAC), further enhanced osteoblast attachment to the surfaces, along with the increase level of intracellular glutathione reserves as a result of cellular uptake of NAC. These results suggest that HEPES coating of titanium surfaces maintained their superhydrophilicity for at least 3 months and resulted in a continuous retention of bioactivity and osteoconductivity similar to freshly prepared surfaces. This coating technology may be useful for preventing biological aging of titanium and delivering biological molecules for synergistic enhancement of bone-titanium integration.

Enhancement of bone-titanium integration profile with UV-photofunctionalized titanium in a gap healing model.

In this study, we tested the potential of UV-photofunctionalized titanium surfaces to overcome compromised bone-titanium integration in a gap healing model. Titanium in rod and disk forms was acid etched and then stored for 4 weeks under dark ambient conditions. Titanium rods with and without UV pretreatment were placed into a rat femur with (contact healing) or without (gap healing) contact with the innate cortical bone. The titanium implants were subjected to a biomechanical push-in test, micro-CT bone morphometry, and surface elemental analysis after 2 weeks of healing. The strength of bone-titanium integration in the gap healing model was one-third of that in the contact healing model. However, UV-treated implants in the gap healing condition produced a strength of bone-titanium integration equivalent to that of untreated implants in the contact healing condition. Bone volume around UV-treated implants was 2- to 3-fold greater than that around the untreated implants in the gap healing model. A bone generation profile drawn along the long axis of the implant exhibited greater contrast between the untreated and UV-treated surfaces in the cortical area than in the bone marrow area. The bone tissue formed on UV-treated implants showed a higher Ca/P ratio than that formed on untreated titanium. The rate of cell proliferation, alkaline phosphatase activity, and calcium deposition in femoral periosteal cells and in bone marrow-derived osteoblasts were greater in cultures on UV-treated titanium disks than in cultures on untreated disks. The UV-enhanced function in periosteal cells was more pronounced when they were co-cultured with bone marrow-derived osteoblasts, indicating a synergistic effect of UV-treated titanium with biological signals from bone marrow-derived osteoblasts. Within the limitation of the model used in this study, UV-photofunctionalized titanium surfaces may overcome the challenging condition of bone-titanium integration without cortical bone support. UV treatment of implants induced marked improvements in the behavior of bone formation and quantity and quality of bone tissue around the implants. These effects may be related to the promoted function of both periosteum- and bone marrow-derived osteogenic cells at the local level around UV-treated titanium surfaces.

N-acetyl cysteine prevents polymethyl methacrylate bone cement extract-induced cell death and functional suppression of rat primary osteoblasts.

This study examines the cytotoxicity of bone cement extract to osteoblasts and the potential detoxification and restoration of osteoblastic function by an antioxidant amino acid, N-acetyl cysteine (NAC). The osteoblastic cells derived from rat femurs were cultured with extract from polymethyl methacrylate (PMMA)-based bone cement. The calcein and ethidium homodimer staining of the cells after 24-h incubation showed that 23.0% of the cells were dead in the culture with bone cement extract, while the addition of 5 mM NAC into the culture reduced the percentage to 4.3%. Annexin V and propidium iodide-based flow cytometric analysis also revealed that the apoptotic cells present at 15.8% in the culture with bone cement extract was reduced to 2.4% in the culture cotreated with bone cement extract and NAC. Severely suppressed alkaline phosphatase activity and matrix mineralization in the culture with bone cement extract (reduced to 10% and 5%, respectively, compared with the control culture) were restored to a normal level when treated with 5 mM NAC. The bone cement extract-induced, downregulated expression of osteoblastic genes, such as alkaline phosphatase, collagen I, and osteocalcin, was also restored to the baseline level by cotreatment with NAC. The data indicated that the addition of NAC into acrylic bone cement extract remarkably ameliorated the cytotoxicity to osteoblasts and restored their phenotype and function to a biologically significant degree, suggesting the potential usefulness of NAC in developing more biocompatible acrylic bone cement.

Ultraviolet light-mediated photofunctionalization of titanium to promote human mesenchymal stem cell migration, attachment, proliferation and differentiation.

Improving the osteoconductive potential of titanium implants has been of continuing interest in the fields of dentistry and orthopedic surgery. This study determined the bioactivity of ultraviolet (UV) light-treated titanium. Human mesenchymal stem cells (MSCs) were cultured on acid-etched microtopographical titanium surfaces with and without 48h pretreatment with UVA (peak wavelength of 360n m) or UVC (peak wavelength of 250 nm). The number of cells that migrated to the UVC-treated surface during the first 3h of incubation was eight times higher than those that migrated to the untreated surface. After 24h of incubation, the number of cells attached to the UVC-treated surface was over three times more than those attached to the untreated surface. On the UVC-treated surface, the cellular spread was expedited with an extensive and intensive expression of the focal adhesion protein vinculin. The cells on the UVC-treated surface exhibited a threefold higher bromodeoxyuridine incorporation, a doubling of the alkaline phosphatase-positive area and the up-regulated expression of bone-related genes, indicating the accelerated proliferation and differentiation. The UVC-treated surface did not adversely affect the viability of the cells. These biological effects were not seen after UVA treatment, despite the generation of superhydrophilicity. Thus, we discovered a novel photofunctionalization of titanium dioxide that substantially enhances its bioactivity in human MSCs. Further studies are required to investigate the universal effectiveness of this surface modification for different titanium-containing materials, with varying chemistries and textures, as well as to understand its significance in enhancing in vivo osteoconductivity.

The effect of UV-photofunctionalization on the time-related bioactivity of titanium and chromium-cobalt alloys.

This study examined the possible changes in the bioactivity of titanium surfaces during their aging and investigated the effect of ultraviolet (UV) light treatment during the age-related change of titanium bioactivity. Rat bone marrow-derived osteoblastic cells were cultured on new titanium disks (immediately after either acid-etching, machining, or sandblasting), 4-week-old disks (stored after processing for 4 weeks in dark ambient conditions), and 4-week-old disks treated with UVA (peak wavelength of 365 nm) or UVC (peak wavelength of 250 nm). During incubation for 24 h, only 50% of the cells were attached to the 4-week-old surfaces as compared to the new surface. UVC treatment of the aged surface increased its cell attachment capacity to a level 50% higher than the new surfaces, whereas UVA treatment had no effect. Proliferation, alkaline phosphatase activity, and mineralization of cells were substantially lower on the 4-week-old surfaces than on the new surfaces, while they were higher on the UVC-treated 4-week-old surfaces as compared to the new surfaces. The age-related impaired bioactivity was found on all titanium topographies as well as on a chromium-cobalt alloy, and was associated with an increased percentage of surface carbon. Although both UVA and UVC treatment converted the 4-week-old titanium surfaces from hydrophobic to superhydrophilic, only UVC treatment effectively reduced the surface carbon to a level equivalent to the new surface. Thus, this study uncovered a time-dependent biological degradation of titanium and chromium-cobalt alloy, and its restoration enabled by UVC phototreatment, which surmounts the innate bioactivity of new surfaces, which is more closely linked to hydrocarbon removal than the induced superhydrophilicity.

Ultraviolet treatment overcomes time-related degrading bioactivity of titanium.

The shelf life of titanium implant products, that is, a possible time-related change of their bioactivity, has rarely been addressed. The objective of this study was to examine the bioactivity of newly processed and aged titanium surfaces and determine whether ultraviolet (UV) light treatment of the titanium surface restores the possible adverse effects of titanium aging. Titanium disks, either acid-etched or sandblasted, were used immediately after processing (fresh surface) or after storing in dark for 4 weeks (aged surface). Some disks were treated with UV light for 48 h after 4 weeks of storage. Albumin adsorbed to the aged surfaces was only 15% of that adsorbed to the fresh surfaces during 2-h incubation, whereas UV-treated aged surfaces adsorbed equivalent amount of albumin to that for the fresh surfaces. During 24-h incubation, the number of human mesenchymal stem cells attached to the aged surfaces was less than half of that for the fresh surfaces, whereas UV treatment of the aged surfaces increased the number three times. Proliferation, alkaline phosphatase activity, and calcium deposition of the cells were substantially lower on the aged surfaces than on the fresh surfaces, while those on the UV-treated aged surfaces were higher than on the fresh surfaces. The strength of bone-implant integration evaluated at week 2 of healing in a rat femur model was reduced to half after 4 weeks of titanium aging, whereas UV treatment of the aged implants increased the strength to the level equivalent to or even higher than the freshly prepared implants. Fresh and UV-treated aged surfaces were superhydrophilic, while the aged surface was hydrophobic. The data suggest that bioactivity of titanium surfaces degrades with time and that UV treatment of the aged surface increases the bioactivity over the level of the freshly prepared surface.

N-acetyl cysteine (NAC)-mediated detoxification and functionalization of poly(methyl methacrylate) bone cement.

Currently used poly(methyl methacrylate) (PMMA)-based bone cement lacks osteoconductivity and induces osteolysis and implant loosening due to its cellular and tissue-toxicity. A high percentage of revision surgery following the use of bone cement has become a significant universal problem. This study determined whether incorporation of the amino acid derivative N-acetyl cysteine (NAC) in bone cement reduces its cytotoxicity and adds osteoconductivity to the material. Biocompatibility and bioactivity of PMMA-based bone cement with or without 25mm NAC incorporation was examined using rat bone marrow-derived osteoblastic cells. Osteoconductive potential of NAC-incorporated bone cement was determined by microCT bone morphometry and implant biomechanical test in the rat model. Generation of free radicals within the polymerizing bone cement was examined using electron spin resonance spectroscopy. Severely compromised viability and completely suppressed phenotypes of osteoblasts on untreated bone cement were restored to the normal level by NAC incorporation. Bone volume formed around 25mm NAC-incorporated bone cement was threefold greater than that around control bone cement. The strength of bone-bone cement integration was 2.2 times greater for NAC-incorporated bone cement. For NAC-incorporated bone cement, the spike of free radical generation ended within 12h, whereas for control bone cement, a peak level lasted for 6 days and a level greater than half the level of the peak was sustained for 20 days. NAC also increased the level of antioxidant glutathione in osteoblasts. These results suggest that incorporation of NAC in PMMA bone cement detoxifies the material by immediate and effective in situ scavenging of free radicals and increasing intracellular antioxidant reserves, and consequently adds osteoconductivity to the material.

N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract.

There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.

Effects of biting on elevation of blood pressure and other physiological responses to stress in rats: biting may reduce allostatic load.

We have investigated how biting modulates some of the physiological changes (blood pressure, core temperature, and chemical mediators in the serum) that are induced by restraint stress. We exposed rats to restraint stress for 60 min. Biting on a wooden stick during restraint significantly suppressed the increase of blood pressure at 30, 45, 60, and 75 min and significantly inhibited the rise in core temperature at 30, 60, 120, and 180 min compared with rats that were restrained but did not bite anything. These differences were visible in infrared thermal images of the restraint-only and restraint-with-biting rats after 60 min. Biochemical analysis revealed that biting significantly suppressed increases of plasma interleukin-1beta, interleukin-6, and leptin and that it significantly suppressed a decrease of thyroid-stimulating hormone. These observations suggest that biting produces an anti-stress effect and that para-functional masticatory activity plays an important role in coping with stressful events.