Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

PURPOSE: Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). METHODS: The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. RESULTS: We found that the recombinant ß-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. CONCLUSIONS: We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

MALT lymphoma of the sublingual gland: A case report with current overview of diagnostic and therapeutic strategies.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is a low-grade B-cell lymphoma. MALT lymphomas involving the sublingual gland are extremely rare. Herein, we report a case of MALT lymphoma of the sublingual gland. Additionally, we discuss challenging diagnostic aspects as well as current treatment strategies.

Novel extraction method using an ISOLUTE PLD+ protein and phospholipid removal column for liquid chromatography-tandem mass spectrometry analysis of 20 psychoactive drugs in postmortem whole blood samples.

A novel sample extraction method using an ISOLUTE PLD+ protein and phospholipid removal column was developed for simultaneous quantification of 20 psychoactive drugs, including antidepressants, antipsychotics, sedative-hypnotics, and amphetamines, in postmortem whole blood samples by liquid chromatography-tandem mass spectrometry. The method showed improvement in extract cleanliness compared with traditional protein precipitation and the QuEChERS extraction method. The method was validated for all analytes; the calibration curves showed good linearity, with r2 values exceeding 0.991. The intra- and interday accuracies and precisions were 87.6-117.5% and 1.0-18.6%, respectively. The recovery efficiencies were in the range of 64.6-96.8%. Matrix effects were observed in the range of 82.6-116.0%. All analytes were stable under different storage conditions. This method was successfully applied in postmortem forensic sample analysis to quantify psychoactive drugs. The method described in the current study will be useful for forensic toxicological investigations.

Melanoma Inhibitory Activity and Melanoma Inhibitory Activity 2 as Novel Immunohistochemical Markers of Oral Epithelial Dysplasia.

Oral potentially malignant disorders are associated with the development of oral squamous cell carcinoma (OSCC). Most OSCCs are diagnosed via histopathology as oral epithelial dysplasia (OED), but the histologic diagnostic criteria remain non-uniform. Accordingly, the establishment of a diagnostic marker to assist in diagnosis could contribute towards cancer prevention. Melanoma inhibitory activity (MIA) and MIA2 are involved in tumor growth, invasion, and lymph node metastasis in various malignancies. The purpose of this study was to clarify the usefulness of MIA and MIA2 as diagnostic markers of oral mucosal lesions. The expression of MIA and MIA2 was analyzed immunohistochemically in 100 specimens (10 specimens with normal oral mucosa (NOM) and 30 specimens each with low-grade epithelial dysplasia (LED), high-grade epithelial dysplasia (HED), and OSCC). Immunohistochemical results were evaluated based on the Allred scoring system. Cytoplasmic expression of MIA and MIA2 increased in the order of LED, HED, and OSCC. All NOM specimens were negative for cytoplasmic expression. Significant differences were observed between the groups (NOM vs. HED, p < 0.05, NOM vs. OSCC, p < 0.001). These results demonstrate that MIA and MIA2 are expressed in the oral mucosa within early neoplastic lesions and suggest that MIA and MIA2 are useful novel immunohistochemical markers for discriminating between normal tissue and OED.

A Newly Created Meso-, Micro-, and Nano-Scale Rough Titanium Surface Promotes Bone-Implant Integration.

Titanium implants are the standard therapeutic option when restoring missing teeth and reconstructing fractured and/or diseased bone. However, in the 30 years since the advent of micro-rough surfaces, titanium's ability to integrate with bone has not improved significantly. We developed a method to create a unique titanium surface with distinct roughness features at meso-, micro-, and nano-scales. We sought to determine the biological ability of the surface and optimize it for better osseointegration. Commercially pure titanium was acid-etched with sulfuric acid at different temperatures (120, 130, 140, and 150 °C). Although only the typical micro-scale compartmental structure was formed during acid-etching at 120 and 130 °C, meso-scale spikes (20-50 µm wide) and nano-scale polymorphic structures as well as micro-scale compartmental structures formed exclusively at 140 and 150 °C. The average surface roughness (Ra) of the three-scale rough surface was 6-12 times greater than that with micro-roughness only, and did not compromise the initial attachment and spreading of osteoblasts despite its considerably increased surface roughness. The new surface promoted osteoblast differentiation and in vivo osseointegration significantly; regression analysis between osteoconductivity and surface variables revealed these effects were highly correlated with the size and density of meso-scale spikes. The overall strength of osseointegration was the greatest when the acid-etching was performed at 140 °C. Thus, we demonstrated that our meso-, micro-, and nano-scale rough titanium surface generates substantially increased osteoconductive and osseointegrative ability over the well-established micro-rough titanium surface. This novel surface is expected to be utilized in dental and various types of orthopedic surgical implants, as well as titanium-based bone engineering scaffolds.

Notch as a Possible Cell Differentiation Factor in Pleomorphic Adenomas.

The expression of Notch in 30 cases of pleomorphic adenoma was examined by immunohistochemistry. Comparing the results of our study with previous literatures, from the partial CK7 expression and substantial Notch expression in ductal epithelial cells as well as the Notch expression in solid tumor nests, it can be inferred that Notch is involved in cell differentiation. CK13 expression was observed in cells undergoing squamous metaplasia and Notch expression was seen in the nucleus of basal and squamous cells. The intense Notch expression in basal cells and weak expression in squamous cells suggests that Notch is involved in the differentiation from basal to squamous cell. Moreover, the loss of nuclear expression on the inner layer would signify that differentiation is about to end or has been terminated. Notch was expressed in the cytoplasm of cartilage cells and in the cell membrane of mucous cells but not in the nucleus indicating that differentiation has been concluded. Notch involvement is suspected in cell differentiation in areas showing ductal structures and squamous metaplasia. In summary, Notch is involved in cell differentiation of ductal cells in PA. Nuclear expression was shown in tumor cells in solid nests and surrounding structures. Moreover, Notch is expressed by basal cells undergoing squamous metaplasia suggesting the participation of Notch in cell differentiation in PA.

Transplantation of cultured dental pulp stem cells into the skeletal muscles ameliorated diabetic polyneuropathy: therapeutic plausibility of freshly isolated and cryopreserved dental pulp stem cells.

INTRODUCTION: Dental pulp stem cells (DPSCs) are mesenchymal stem cells located in dental pulp and are thought to be a potential source for cell therapy since DPSCs can be easily obtained from teeth extracted for orthodontic reasons. Obtained DPSCs can be cryopreserved until necessary and thawed and expanded when needed. The aim of this study is to evaluate the therapeutic potential of DPSC transplantation for diabetic polyneuropathy. METHODS: DPSCs isolated from the dental pulp of extracted incisors of Sprague-Dawley rats were partly frozen in a -80 °C freezer for 6 months. Cultured DPSCs were transplanted into the unilateral hindlimb skeletal muscles 8 weeks after streptozotocine injection and the effects of DPSC transplantation were evaluated 4 weeks after the transplantation. RESULTS: Transplantation of DPSCs significantly improved the impaired sciatic nerve blood flow, sciatic motor/sensory nerve conduction velocity, capillary number to muscle fiber ratio and intra-epidermal nerve fiber density in the transplanted side of diabetic rats. Cryopreservation of DPSCs did not impair their proliferative or differential ability. The transplantation of cryopreserved DPSCs ameliorated sciatic nerve blood flow and sciatic nerve conduction velocity as well as freshly isolated DPSCs. CONCLUSIONS: We demonstrated the effectiveness of DPSC transplantation for diabetic polyneuropathy even when using cryopreserved DPSCs, suggesting that the transplantation of DPSCs could be a promising tool for the treatment of diabetic neuropathy.

Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge.

Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 10(7) BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 10(6) BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 µg of recombinant human BMP-2 (rhBMP-2) or 1.8 × 10(7) empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development.

Role of photofunctionalization in mitigating impaired osseointegration associated with type 2 diabetes in rats.

PURPOSE: Peri-implant osteogenesis is reported to be impaired in patients with diabetes. The current study tested the hypothesis that ultraviolet (UV) treatment of titanium, or photofunctionalization, is able to mitigate the impaired osseointegration associated with type 2 diabetes. MATERIALS AND METHODS: Untreated and photofunctionalized titanium implants were placed into the femurs of genetically modified rats with a close phenotypic resemblance to human type 2 diabetes, as characterized by late-onset hyperglycemia and obesity. Implants were photofunctionalized with UV light for 15 minutes immediately before placement. The strength of osseointegration was evaluated using a biomechanical push-in test, and the tissue-implant interface was examined using scanning electron microscopy and energy-dispersive spectroscopy. RESULTS: Photofunctionalization converted implants from hydrophobic to superhydrophilic. Photofunctionalization-induced hemophilicity was also confirmed during surgery. The strength of osseointegration of photofunctionalized implants was significantly greater than that of untreated implants, by 1.8 and 3 times, at weeks 2 and 4 of healing, respectively. Osseointegration of photofunctionalized implants in diabetic animals was even stronger than that of untreated implants placed in normal animals throughout the healing period. Photofunctionalized implants placed in diabetic rats were extensively covered with calcium- and phosphorus-rich tissue that masked the titanium signal. CONCLUSION: Photofunctionalization accelerated and enhanced levels of osseointegration and overcame impaired osseointegration in a rat model of type 2 diabetes. Further prospective studies are warranted to establish the clinical efficacy of photofunctionalization in patients with diabetes.

Wnt signaling as a possible promoting factor of cell differentiation in pleomorphic adenomas.

There are well known that Wnt signaling was some roles of cell differentiation at the development tissues, especially the oral and maxillofacial regions of some developmental stages. Therefore, to determine Wnt signaling in the pleomorphic adenoma tissues, we examined. The expression of Wnt1 and ß-catenin as well as the distribution of various cytoskeletal proteins CK7 and CK13 was examined in 30 cases of pleomorphic adenoma by immunohistochemistry. Wnt1 was detected in almost all tumor cells. The peripheral columnar cells in squamous metaplasia and small cuboidal cells in duct-like structures were strongly positive to Wnt1. Although ß-catenin was clearly localized on the cell membrane of tumor cells, nuclear translocation was observed in small cuboidal cells and in some basaloid cells. The immunofluorescent staining pattern of Wnt1 and CK7 as well as Wnt1 and CK13 was consistent with IHC results. Thus, in pleomorphic adenoma, Wnt is involved in tumor cell differentiation of peripheral columnar cells forming solid nests and small peripheral columnar cells forming duct-like structures. Moreover, among the three currently known Wnt pathways, ß-catenin is the suggested pathway working during cell differentiation. Furthermore, peripheral columnar cells in solid tumor nests and in squamous metaplasia are governed by another Wnt pathway other than ß-catenin. Therefore, Wnt signaling through ß-catenin pathway may be involved in the 'mixed' differentiation characteristic of pleomorphic adenoma although another pathway may also be possibly working in other parts of the tumor tissue.

Gelatin powders accelerate the resorption of calcium phosphate cement and improve healing in the alveolar ridge.

The aim of this study was to show the effectiveness of combining calcium phosphate cement and gelatin powders to promote bone regeneration in the canine mandible. We mixed gelatin powders with calcium phosphate cement to create a macroporous composite. In four beagle dogs, two saddle-type bone defects were created on each side of the mandible, and calcium phosphate cement alone or calcium phosphate cement containing composite gelatin powders was implanted in each of the defects. After a healing period of six months, mandibles were removed for µCT and histological analyses. The µCT and histological analyses showed that at experimental sites at which calcium phosphate cement alone had been placed new bone had formed only around the periphery of the residual calcium phosphate cement and that there had been little or no ingrowth into the calcium phosphate cement. On the other hand, at experimental sites at which calcium phosphate cement containing composite gelatin powders had been placed, we observed regenerated new bone in the interior of the residual calcium phosphate cement as well as around its periphery. The amount of resorption of calcium phosphate cement and bone regeneration depended on the mixing ratio of gelatin powders to calcium phosphate cement. New bone replacement was significantly better in the sites treated with calcium phosphate cement containing composite gelatin powders than in those treated with calcium phosphate cement alone.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

Evaluation of guided bone regeneration with poly(lactic acid-co-glycolic acid-co-ε-caprolactone) porous membrane in lateral bone defects of the canine mandible.

PURPOSE: The aim of this study was to qualitatively evaluate a poly(lactic acid-co-glycolic acid-co-ε-caprolactone) (PLGC) membrane as a barrier for guided bone regeneration in the canine mandible and to compare it to a nonresorbable polytetrafluoroethylene (PTFE) membrane. MATERIALS AND METHODS: Two wedge-shaped bone defects were created bilaterally in the mandibles of 12 beagle dogs. The bone defects in the left mandible were divided into three groups and treated as follows: PLGC membrane alone, PLGC membrane plus autogenous cortical bone chips, and titanium-reinforced expanded PTFE (TR-PTFE) membrane. The bone defects in the right mandible of each animal were left without membranes as a control. Computed tomography (CT) was performed at 3 and 6 months postoperative to evaluate bone regeneration. After a healing period of 6 months, the mandibles were removed en bloc for micro-CT and histologic analyses. RESULTS: CT analyses at 3 and 6 months showed that there was significantly more bone augmentation at all experimental sites than at the control sites. The volume of bone at defect sites covered with TR-PTFE was significantly greater than at defect sites covered with PLGC membrane with or without autogenous cortical bone. Micro-CT measurements showed that the volume of new bone formed at sites covered with TR-PTFE was significantly greater than at sites covered with PLGC membrane. However, the density of new bone was significantly higher at sites covered with PLGC membrane, with or without cortical bone, than at sites covered with TR-PTFE. Histologic analysis verified the presence of well-vascularized loose connective tissue in the pores of the PLGC membrane. CONCLUSIONS: Compared to TR-PTFE, the macroporous bioresorbable PLGC membrane did not significantly increase the amount of new bone in defect sites, but it facilitated the regeneration of mature bone.

Alveolar bone regeneration using poly-(lactic acid-co-glycolic acid-co-ε-caprolactone) porous membrane with collagen sponge containing basic fibroblast growth factor: an experimental study in the dog.

The aim of this study was to evaluate the effects of combining porous poly-lactic acid-co-glycolic acid-co-ε-caprolactone (PLGC) as a barrier membrane and collagen sponge containing basic fibroblast growth factor (bFGF) to promote bone regeneration in the canine mandible. In six beagle dogs, two lateral bone defects per side were created in the mandible. The lateral bone defects on the left side were treated with a PLGC membrane plus a collagen sponge containing bFGF. In half of these, the collagen sponge contained 50 µg of bFGF. In the other half, it contained 250 µg of bFGF. As a control, we treated the right-side bone defects in each animal with the same PLGC membrane but with a collagen sponge containing phosphate buffered saline. Computed tomography (CT) images were recorded at 3 and 6 months post-op to evaluate regeneration of the bone defects. After a healing period of 6 months, whole mandibles were removed for micro-CT and histological analyses. The post-op CT images showed that more bone had formed at all experimental sites than at control sites. At 3 months post-op, the volume of bone at defect sites covered with PLGC membrane plus 250 µg of bFGF was significantly greater than it was at defect sites covered with PLGC membrane plus 50 µg of bFGF. At 6 months post-op, however, this difference was smaller and not statistically significant. Micro-CT measurement showed that the volume of new bone regenerated at bone-defect sites, covered with PLGC membrane plus bFGF, was significantly greater than that of control sites. However, the presence or absence of bFGF in the collagen sponge did not significantly affect the bone density of new bone. These results suggest that the macroporous bioresorbable PLGC membrane plus collagen sponge containing bFGF effectively facilitates healing in GBR procedures.

TiO2 micro-nano-hybrid surface to alleviate biological aging of UV-photofunctionalized titanium.

Bioactivity and osteoconductivity of titanium degrade over time after surface processing. This time-dependent degradation is substantial and defined as the biological aging of titanium. UV treatment has shown to reactivate the aged surfaces, a process known as photofunctionalization. This study determined whether there is a difference in the behavior of biological aging for titanium with micro-nano-hybrid topography and titanium with microtopography alone, following functionalization. Titanium disks were acid etched to create micropits on the surface. Micro-nano-hybrid surfaces were created by depositioning 300-nm diameter TiO(2) nodules onto the micropits using a previously established self-assembly protocol. These disks were stored for 8 weeks in the dark to allow sufficient aging, then treated with UV light for 48 hours. Rat bone marrow-derived osteoblasts were cultured on fresh disks (immediately after UV treatment), 3-day-old disks (disks stored for 3 days after UV treatment), and 7-day- old disks. The rates of cell attachment, spread, proliferation, and levels of alkaline phosphatase activity, and calcium deposition were reduced by 30%-50% on micropit surfaces, depending on the age of the titanium. In contrast, 7-day-old hybrid surfaces maintained equivalent levels of bioactivity compared with the fresh surfaces. Both micropit and micro-nano-hybrid surfaces were superhydrophilic immediately after UV treatment. However, after 7 days, the micro-nano- hybrid surfaces became hydrorepellent, while the micropit surfaces remained hydrophilic. The sustained bioactivity levels of the micro-nano-hybrid surfaces were nullified by treating these surfaces with Cl(-)anions. A thin TiO(2) coating on the micropit surface without the formation of nanonodules did not result in the prevention or alleviation of the time-dependent decrease in biological activity. In conclusion, the micro-nano-hybrid titanium surfaces may slow the rate of time-dependent degradation of titanium bioactivity after UV photofunctionalization compared with titanium surfaces with microtopography alone. This antibiological aging effect was largely regulated by its sustained electropositivity uniquely conferred in TiO(2) nanonodules, and was independent of the degree of hydrophilicity. These results demonstrate the potential usefulness of these hybrid surfaces to effectively utilize the benefits of UV photofunctionalization and provide a model to explore the mechanisms underlying antibiological aging properties.

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.

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.

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.

Biomechanical properties of jaw periosteum-derived mineralized culture on different titanium topography.

PURPOSE: This study evaluated the biomechanical properties of periosteum-derived mineralized culture on different surface topographies of titanium. MATERIALS AND METHODS: Titanium surfaces modified by machining or by acid etching were analyzed using scanning electron microscopy (SEM). Rat mandibular periosteum-derived cells were cultured on either of the titanium surfaces. Cell proliferation was evaluated by cell counts, and gene expression was analyzed using a reverse-transcriptase polymerase chain reaction. Alkaline phosphatase (ALP) stain assay was employed to evaluate osteoblastic activity. Matrix mineralization was examined via von Kossa stain assay, total calcium deposition, and SEM. The hardness and elastic modulus of mineralized cultures were measured using a nano-indenter. RESULTS: The machined surface demonstrated a flat topographic configuration, while the acid-etched surface revealed a uniform micron-scale roughness. Both cell density and ALP activity were significantly higher on the machined surface than on the acid-etched surface. The expression of bone-related genes was up-regulated or enhanced on the acid-etched surface compared to the machined surface. Von Kossa stain showed significantly greater positive areas for the machined surface compared to the acid-etched surface, while total calcium deposition was statistically similar. Mineralized culture on the acid-etched surface was characterized by denser calcium deposition, more mature collagen deposition on the superficial layer, and larger and denser globular matrices inside the matrix than the culture on the machined surface. The mineralized matrix on the acid-etched surface was two times harder than on the machined surface, whereas the elastic modulus was comparable between the two surfaces. CONCLUSIONS: The design of this study can be used as a model to evaluate the effect of implant surface topography on the biomechanical properties of periosteum-derived mineralized culture. The results suggest that mandibular periosteal cells respond to different titanium surface topographies differently enough to produce mineralized matrices with different biomechanical qualities.