Evaluation of osteoblastic cell behavior upon culture on titanium substrates photo-functionalized by vacuum ultra-violet treatment.

Photo-functionalization of titanium orthopedic/prosthetic implants using ultraviolet illumination is known to improve osteogenesis. Therefore, in this study, we aimed to examine the influence of vacuum ultraviolet (VUV)-treated titanium surfaces on osteoblast cell adhesion, activity, and differentiation. Osteoblastic cells were cultured on titanium substrates treated with various VUV treatment conditions (0, 6.2, 18.7, and 37.4 J/cm2) and their behavior was evaluated. The results revealed that cell adhesion was increased whereas cell activity and differentiation ability were decreased upon cell culture on VUV-treated substrates. In particular, cell activity and differentiation ability were dramatically suppressed with 18.7 J/cm2 VUV irradiation. Within the limitations of this cell-based experiment, we clarified the VUV treatment conditions in which cell adhesion was improved but cell activity and differentiation ability were suppressed. These results indicate that VUV-treatment can be used to influence cell growth properties and can be used to accelerate or suppress cell differentiation on implant substrates.

Evaluation of humeral head cartilage using magnetic resonance imaging T1 rho mapping for patients with small-to-medium rotator cuff tears: A pilot study.

BACKGROUND: It is unclear whether smaller rotator cuff tears cause cartilage degeneration. This study was designed to detect early humeral head cartilage degeneration in patients with small-to-medium cuff tears using magnetic-resonance-imaging T1 rho mapping. METHODS: Five male and 5 female volunteers without shoulder symptoms (control group) and 5 male and 5 female patients with small-to-medium (<3 cm) rotator cuff tears underwent 3.0-T magnetic resonance imaging of a single shoulder. T1 rho values of the humeral head cartilage were measured and analyzed. RESULTS: The total mean T1 rho value was 40.4 ± 3.4 ms for the control group and 45.0 ± 5.3 ms for the patient group. In the control group, the T1 rho values in the inferior articular cartilage were significantly higher than those in the superior and middle articular cartilage. In the patient group, there was no significant difference between all regions. A comparison between the patient and control groups showed that the mean T1 rho values in the superior-to-middle articular cartilage were significantly higher for the patient group than for the control group. However, in the inferior articular cartilage, there was no significant difference between both groups. CONCLUSIONS: This study showed the possibility of early cartilage degenerative changes in the superior-to-middle humeral head articular cartilage of patients with small-to-medium rotator cuff tears.

In vivo kinematic analysis of the glenohumeral joint during dynamic full axial rotation and scapular plane full abduction in healthy shoulders.

PURPOSE: The purpose of this study was to evaluate the kinematics of healthy shoulders during dynamic full axial rotation and scapular plane full abduction using three-dimensional (3D)-to-two-dimensional (2D) model-to-image registration techniques. METHODS: Dynamic glenohumeral kinematics during axial rotation and scapular plane abduction were analysed in 10 healthy participants. Continuous radiographic images of axial rotation and scapular plane abduction were taken using a flat panel radiographic detector. The participants received a computed tomography scan to generate virtual digitally reconstructed radiographs. The density-based digitally reconstructed radiographs were then compared with the serial radiographic images acquired using image correlations. These 3D-to-2D model-to-image registration techniques determined the 3D positions and orientations of the humerus and scapula during dynamic full axial rotation and scapular plane full abduction. RESULTS: The humeral head centre translated an average of 2.5 ± 3.1 mm posteriorly, and 1.4 ± 1.0 mm superiorly in the early phase, then an average of 2.0 ± 0.8 mm inferiorly in the late phase during external rotation motion. The glenohumeral external rotation angle had a significant effect on the anterior/posterior (A/P) and superior/inferior (S/I) translation of the humeral head centre (both p < 0.05). 33.6 ± 15.6° of glenohumeral external rotation occurred during scapular plane abduction. The humeral head centre translated an average of 0.6 ± 0.9 mm superiorly in the early phase, then 1.7 ± 2.6 mm inferiorly in the late phase, and translated an average of 0.4 ± 0.5 mm medially in the early phase, then 1.6 ± 1.0 mm laterally in the late phase during scapular plane abduction. The humeral abduction angle had a significant effect on the S/I and lateral/medial (L/M) translation of the humeral head centre (both p < 0.05). CONCLUSION: This study investigated 3D translations of the humerus relative to the scapula: during scapular plane full abduction, the humerus rotated 33.6° externally relative to the scapula, and during external rotation motion in the adducted position, the humeral head centre translated an average of 2.5 mm posteriorly. Kinematic data will provide important insights into evaluating the kinematics of pathological shoulders. For clinical relevance, quantitative assessment of dynamic healthy shoulder kinematics might be a physiological indicator for the assessment of pathological shoulders.

Stress fracture of the navicular bone in a patient with cerebral palsy: a case report.

A 14-year-old girl with cerebral palsy (spastic diplegia) underwent examination due to a chief complaint of right foot pain, and was diagnosed with a stress fracture of the central one third of the navicular bone. The fracture was considered to have developed due to repeated loading on the navicular bone as a result of an equinus gait.Therefore, she underwent osteosynthesis and Achilles tendon lengthening to correct the equinus deformity. Following our review of the current literature, we did not identify any reports of stress fracture of the navicular bone in cerebral palsy. We believe that in cases where cerebral palsy patients with paralytic equinus complain of foot pain, the possibility of stress fracture of the navicular bone should be considered.

[Retrospective analysis of clinical and pathological factors that influence patient survival in salivary gland cancer - could chemotherapy improve survival ?].

The purpose of this retrospective study was to evaluate the prognostic factors in salivary gland cancer using a multivariate analysis. In total, 45 consecutive patients who underwent planned radical resection for salivary gland cancer between 1985 and 2010 were analyzed. Univariate and multivariate analyses were performed to analyze the clinical and pathological factors that influence patient survival in salivary gland cancer(tumor location, T and N classification, histological grade, excision margin status, postoperative radiotherapy, and postoperative adjuvant chemotherapy). The results of the multivariate analysis showed that T classification(odds ratio[OR]: 2.93, 95%confidence interval[CI]: 1.47-5.82), and excision margin status(OR: 4.86, 95% CI: 1.76-13.44)had a significant impact on tumor-free survival time, suggesting that both are important prognostic factors in salivary gland cancer. Moreover, postoperative adjuvant chemotherapy showed a tendency to improve tumor free survival time(OR: 0.28, 95% CI: 0.07-1.11), thereby indicating its potential in improving prognosis in patients with salivary gland cancer.

Vital staining with iodine solution in oral cancer: iodine infiltration, cell proliferation, and glucose transporter 1.

BACKGROUND: Vital staining with iodine solution has been used to distinguish dysplastic/malignant oral epithelium from normal mucosa. However, little is known about its critical mechanism. The purpose of this study was to visualize how iodine infiltrates the oral epithelium and reacts with glycogen. In addition, we tested the hypothesis that higher cell proliferation requires increased energy consumption, and consequently exhausted glycogen may lead to a failure to be stained by iodine solution. METHODS: Fifteen frozen tissue samples of iodine-stained and -unstained mucosa were obtained from 15 cases of oral squamous cell carcinoma (OSCC). Serial frozen sections were cut and examined with hematoxylin and eosin and periodic acid-Schiff methods and immunohistochemical staining for p53, Ki67 and glucose transporter 1 (GLUT 1). RESULTS: Iodine solution was able to penetrate normal epithelium to a maximum depth neighboring the parabasal layer, but iodine-stained areas were completely consistent with glycogen distribution only in the upper superficial layer. Iodine-negative epithelium presented significantly higher immunoreactions for P53 and GLUT 1 in basal, parabasal, and superficial layers, respectively, whereas the reaction for Ki67 in the superficial layer was higher than that in iodine-positive epithelium (Wilcoxon signed-rank test, P < 0.05). CONCLUSIONS: Iodine infiltrated and reacted with glycogen mainly in the upper superficial layer of the nonkeratinized epithelium. Both histological and molecular margins can be confirmed by iodine vital staining in OSCC. It is also suggested that high cell proliferation induced elevated glycolysis, resulting in an intraepithelial glycogen degradation and consequent failure to be stained by iodine solution.

Impact of particulate microplastics generated from polyethylene terephthalate on gut pathology and immune microenvironments.

Environmental microplastics have emerged as a critical issue in maintaining the planetary ecosystem. In this study, we generated particulate microplastics from polyethylene terephthalate (PM-PET) and investigated their impact in the gut by using mouse models and implementing histological examinations, as well as multi-omics analysis for colonic immune cells and microbiota. As a result, histological approaches showed that chronic and physiological low dose exposure of PM-PET did not affect intestinal pathology and mucin barriers, respectively. Moreover, immunohistochemical analysis demonstrated that the numbers of T cells, B cells, macrophages, and granulocytes were not affected by the exposure to PM-PET. However, RNA-seq analysis revealed that PM-PET had a substantial impact on the transcriptome in gut immune cells and their metabolisms, while 16s rRNA metagenomic analysis showed that the composition of microbiota was modestly affected. These results suggest an unexpected role played by the PM-PET in affecting gut immune homeostasis without detectable inflammation.

Protocol for acquiring samples to assess the impact of microplastics on immune microenvironments in the mouse intestine.

Environmental nano- and microplastics (NMPs) pose serious environmental issues, yet there is no established technique to assess their impact on health through oral ingestion. Here, we present a protocol to assess the impact of NMPs in the intestinal immune microenvironments by employing chronic exposure to NMPs in a mouse model. We describe steps for administration of NMPs, feces and tissue collection, and colonic gut digestion. We then detail procedures for isolation of intestinal immune cells and RNA isolation. For complete details on the use and execution of this protocol, please refer to Harusato et al.1.

Reverse Obliquity Intertrochanteric Fracture Treated with a Dynamic Distal Locked Intramedullary Hip Nail.

INTRODUCTION: The aim of this study was to describe the clinical and radiological results of reverse obliquity intertrochanteric fractures treated with dynamic distal locked intramedullary hip nails. MATERIALS AND METHODS: Patients with a reverse obliquity intertrochanteric fracture (AO/OTA type 31 A3.1 or A3.3) underwent surgical treatment with a dynamic distal locked intramedullary hip nail between August 2017 and September 2020. Fracture type, reduction quality, change in the position of the distal bone fragment, bone union, complications, and walking ability were evaluated. RESULTS: Ten patients with reverse obliquity intertrochanteric fractures (seven females and three males) underwent dynamic distal locked intramedullary hip nailing. The mean age was 72.0 ± 18.9 years. Two patients were classi fied as having A3.1 fractures; eight patients were classified as having A3.3 fractures. Anatomical reduction was achieved in six patients, medial displacement remained in three patients, and lateral displacement remained in one patient postoperatively. In 9 out of 10 patients, the center of the proximal end of the distal bone fragment had moved laterally between the time immediately after surgery and the time of the final evaluation. Bone union was obtained uneventfully without any additional treatments in all patients. There were no complications such as infection or implant failure. Walking ability decreased in four patients. CONCLUSION: In all patients, bone union was achieved due to good contact of the medial cortices or the migration of the distal bone fragment into the proximal bone fragment. This procedure can be an option for treating reverse obliquity intertrochanteric fractures.

Surface fractures in pre-crystallized and crystallized zirconia-containing lithium silicate glass-ceramics generated in ultrasonic vibration-assisted machining.

Machining-induced surface fractures in ceramic restorations is a long-standing problem in dentistry, affecting the restorations' functionality and reliability. This study approached a novel ultrasonic vibration-assisted machining technique to zirconia-containing lithium silicate glass-ceramics (ZLS) and characterized its induced surface fracture topographies and morphologies to understand the microstructure-property-processing relations. The materials were processed using a digitally controlled ultrasonic milling machine at a harmonic vibration frequency with different amplitudes. Machining-induced surface fracture topographies were measured with a 3D white light optical profilometer using the arithmetic mean, peak and valley, and maximum heights, as well as the kurtosis and skewness height distributions, and the texture aspect ratios. Fracture morphologies were analysed using scanning electron microscopy (SEM). The surface fracture topographies were significantly dependent on the material microstructure, the mechanical properties, and the ultrasonic machining vibration amplitudes. Larger scale fractures with higher arithmetic mean, peak and valley heights, and kurtosis and skewness height distributions were induced in higher brittleness indexed pre-crystallized ZLS than lower indexed crystallized ZLS by conventional machining. Conchoidal fractures occurred in pre-crystallized ZLS while microcracks were found in crystallized state although brittle fractures mixed with localized ductile flow deformations dominated all machined ZLS surfaces. Ultrasonic machining at an ideal vibration amplitude resulted in more ductile removal, reducing fractured-induced peaks and valleys for both materials than conventional processing. This research demonstrates the microstructure-property-processing interdependence for ZLS materials and the novel machining technique to be superior to current processing, reducing fractures in the materials and potentially advancing dental CAD/CAM techniques.

Friction control of elastic materials on glass by means of textured surfaces.

To investigate the friction behaviors of elastomer and polyacetal writing tips sliding on various textured glass surfaces, the influences of the pitch size and height of sub-millimeter to millimeter sized texture on friction were examined via reciprocating friction tests. The friction coefficients of each writing tip could be systematically varied by changing the pitch and height of the texture. These changes in friction were based on the relationship between the convex-concave shapes and the contact parts of the writing tip, and hence, influence the adhesive, abrasive, and deformation frictions. By inducing a surface texture with a pitch smaller than the contact area of the writing tip, the friction coefficient could be reduced effectively. By inducing a surface texture with a larger height, the friction coefficient of the elastomer could be increased due to deformation friction. These behaviors indicate the possibility of controlling the friction by changing the parameters such as the pitch and height of the textured glass surfaces.

Reversible Thermo-Responsive Valve for Microfluidic Paper-Based Analytical Devices.

Fluid control on a paper channel is necessary for analysis with multiple reagents, such as enzyme-linked immunosorbent assay (ELISA) in microfluidic paper-based analytical devices (µPADs). In this study, a thermo-responsive valve was fabricated by polymerizing N-isopropylacrylamide on a PVDF porous membrane by plasma-induced graft polymerization. The polymerized membrane was observed by scanning electron microscopy (SEM), and it was confirmed that more pores were closed at temperatures below 32 °C and more pores were opened at temperatures above 32 °C. Valve permeability tests confirmed that the proposed polymerized membrane was impermeable to water and proteins at temperatures below 32 °C and permeable to water at temperatures above 32 °C. The valve could also be reversibly and repeatedly opened and closed by changing the temperature near 32 °C. These results suggest that plasma-induced graft polymerization may be used to produce thermo-responsive valves that can be opened and closed without subsequent loss of performance. These results indicate that the thermo-responsive valve fabricated by plasma-induced graft polymerization could potentially be applied to ELISA with µPADs.

Fabrication of Three-Dimensionally Deformable Metal Structures Using Precision Electroforming.

It is difficult to fabricate three-dimensional structures using semiconductor-process technology, because it is based on two-dimensional layered structure fabrication and the etching of thin films. In this study, we fabricated metal structures that can be dynamically deformed from two-dimensional to three-dimensional shapes by combining patterning using photolithography with electroforming technology. First, a resist structure was formed on a Cu substrate. Then, using a Ni sulfamate electroforming bath, a Ni structure was formed by electroforming the fabricated resist structure. Finally, the resist structure was removed to release the Ni structure fabricated on the substrate, and electroforming was used to Au-plate the entire surface. Scanning-electron microscopy revealed that the structure presented a high aspect ratio (thickness/resist width = 3.5), and metal structures could be fabricated without defects across the entire surface, including a high aspect ratio. The metallic structures had an average film thickness of 12.9 µm with σ = 0.49 µm, hardness of 600 HV, and slit width of 7.9 µm with σ = 0.25 µm. This microfabrication enables the fabrication of metal structures that deform dynamically in response to hydrodynamic forces in liquid and can be applied to fields such as environmental science, agriculture, and medicine.

Prevalence of and risk factors for postoperative complications after lower third molar extraction: A multicenter prospective observational study in Japan.

Lower third molar extraction is the most common surgical treatment among routine dental and oral surgical procedures. while the surgical procedures for lower third molar extraction are well established, the difficulty of tooth extraction and the frequency of postoperative complications differ depending on the patient's background. To establish a management protocol for the lower third molars, the prevalence of and risk factors for postoperative complications after lower third molar extraction were investigated in a large number of Japanese patients in a multicenter prospective study. During 6 consecutive months in 2020, 1826 lower third molar extractions were performed at the 20 participating institutions. The medical records of the patients were reviewed, and relevant data were extracted. The prevalence of and risk factors for postoperative complications were analyzed. The prevalence of postoperative complications after lower third molar extraction was 10.0%. Multivariate analysis indicated that age (≤32 vs >32, odds ratio [OR]: 1.428, 95% confidence interval [95% CI]: 1.040-1.962, P < .05), the radiographic anatomical relationship between the tooth roots and mandibular canal (overlapping of the roots and canal vs no close anatomical relationship between the roots and the superior border of the canal, OR: 2.078, 95% CI: 1.333-3.238, P < .01; overlapping of the roots and canal vs roots impinging on the superior border of the canal, OR: 1.599, 95% CI: 1.050-2.435, P < .05), and impaction depth according to the Pell and Gregory classification (position C vs position A, OR: 3.7622, 95% CI: 2.079-6.310, P < .001; position C vs position B, OR: 2.574, 95% CI: 1.574-4.210, P < .001) are significant independent risk factors for postoperative complications after lower third molar extraction. These results suggested that higher age and a deeply impacted tooth might be significant independent risk factors for postoperative complications after lower third molar extraction.

Detection of cancer cells in whole blood using a dynamic deformable microfilter and a nucleic acid aptamer.

Cancer cell count in the blood of cancer patients is extremely low. If these cells are easily detectable, cancer diagnosis may be possible by simply using a blood test, thus reducing patient burden. This study aimed to develop a cancer detection device by combining a microfilter that can be dynamically deformed and a nucleic acid aptamer that has a specific binding ability to cancer cells for easy detection. The cancer detection device was fabricated by photolithography, electroforming, and three-dimensional printing. The cancer cell detection ability of the fabricated device was evaluated using 1 mL of blood samples spiked with different concentrations of cancer cells. The lowest concentration of cancer cells in the blood was 5 cancer cells/1 mL blood. The fabricated microfilters specifically detected cancer cells in the blood successfully at exceedingly low concentrations. Moreover, the cancer detection experiment results using human whole blood revealed that cancer detection could be performed with higher accuracy using the fabricated cancer detection device compared to pre-existing cancer detection equipment (e.g., CellSearch system, Veridex). These findings provide important insights into the use of cancer cells in the blood as a diagnostic approach for cancer.

A dynamically deformable microfilter for selective separation of specific substances in microfluidics.

To study an environmental or biological solution, it is essential to separate its constituents. In this study, a 3D-deformable dynamic microfilter was developed to selectively separate the target substance from a solution. This microfilter is a fine metallic nickel structure fabricated using photolithography and electroplating techniques. It is gold-coated across its entire surface with multiple slits of 10-20 µm in width. Its two-dimensional shape is deformed into a three-dimensional shape when used for fluid separation due to hydrodynamic forces. By adjusting the pressure applied to the microfilter, the size of the gap created by deformation can be changed. To effectively isolate the target substance, the relationship between the solution flow rate and the extent of microfilter deformation was investigated. The filtration experiments demonstrated the microfilter's ability to isolate the target substance with elastic deformation without undergoing plastic deformation. Additionally, modification of the microfilter surface with nucleic acid aptamers resulted in the selective isolation of the target cell, which further demonstrates the potential application of microfilters in the isolation of specific components of heterogeneous solutions.

Spectral analysis of field potential recordings by deep brain stimulation electrode for localization of subthalamic nucleus in patients with Parkinson's disease.

AIMS: Spectral analysis of local field potential (LFP) recorded by deep brain stimulation (DBS) electrode around the subthalamic nucleus (STN) in patients with Parkinson's disease was performed. METHODS: The borders of the STN were determined by microelectrode recording. The most eligible trajectory for the sensorimotor area of the STN was used for LFP recording while advancing the DBS electrode. RESULTS: The low-frequency LFP power (theta- to beta-band) increased from a few millimeters above the dorsal border of the STN defined by microelectrode recording; however, the low-frequency power kept the same level beyond the ventral border of the STN. Only high beta-power showed close correlation to the dorsal and ventral borders of the STN. CONCLUSIONS: A spectral power analysis of LFP recording by DBS electrode helps with the final confirmation of the dorsal and ventral borders of the STN of Parkinson's disease in DBS implantation surgery.

Evaluation of skills in arthroscopic training based on trajectory and force data.

Objective evaluation of surgical skills is essential for an arthroscopic training system. We asked whether a quantitative assessment of arthroscopic skills using scores, time to completion, instrument tip trajectory data, and force data was valid. We presumed more experienced surgeons would perform better on a simulated arthroscopic procedure than novices, therefore validating the quantitative assessment. Surgical trainees (n = 12), orthopaedic residents (n = 12), and experienced arthroscopic surgeons (n = 6) were tested on a Sawbones knee simulator. Subjects performed a joint inspection and probing task and a partial meniscectomy task. The trajectory data were measured using an electromagnetic motion tracking system and the force data were measured using a force sensor. The experienced group performed both tasks with higher scores and more quickly than the less experienced groups. The path length of the probe and the scissors was substantially shorter and the probe velocity was considerably faster in the experienced group. The trainee group applied substantially stronger forces to the joint during the joint inspection and probing task. Our data suggest a performance assessment using an electromagnetic motion tracking system and a force sensor provides an objective means of evaluating surgical skills in an arthroscopic training system.

Constitutive release of cytokines by human oral keratinocytes in an organotypic culture.

PURPOSE: The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME. MATERIALS AND METHODS: Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay. RESULTS: Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P < .001, r = 0.743). CONCLUSION: These results suggest that the increasing detectable levels of VEGF associated with the increasing number of viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME.

Histological evaluation of tendon formation using a scaffold-free three-dimensional-bioprinted construct of human dermal fibroblasts under in vitro static tensile culture.

INTRODUCTION: Tendon tissue engineering requires scaffold-free techniques for safe and long-term clinical applications and to explore alternative cell sources to tenocytes. Therefore, we histologically assessed tendon formation in a scaffold-free Bio-three-dimensional (3D) construct developed from normal human dermal fibroblasts (NHDFs) using our Bio-3D printer system under tensile culture in vitro. METHODS: Scaffold-free ring-like tissues were constructed from 120 multicellular spheroids comprising NHDFs using a bio-3D printer. Ring-like tissues were cultured in vitro under static tensile-loading with or without in-house tensile devices (tension-loaded and tension-free groups), with increases in tensile strength applied weekly to the tensile-loaded group. After a 4 or 8-week culture on the device, we evaluated histological findings according to tendon-maturing score and immunohistological findings of the middle portion of the tissues for both groups (n = 4, respectively). RESULTS: Histology of the tension-loaded group revealed longitudinally aligned collagen fibers with increased collagen deposition and spindle-shaped cells with prolonged culture. By contrast, the tension-free group showed no organized cell arrangement or collagen fiber structure. Additionally, the tension-loaded group showed a significantly improved tendon-maturing score as compared with that for the tension-free group at week 8. Moreover, immunohistochemistry revealed tenascin C distribution with a parallel arrangement in the tensile-loading direction at week 8 in the tension-loaded group, which exhibited stronger scleraxis-staining intensity than that observed in the tension-free group at weeks 4 and 8. CONCLUSIONS: The NHDF-generated scaffold-free Bio-3D construct underwent remodeling and formed tendon-like structures under tensile culture in vitro.