The Influence of Tacrolimus on Cellular Morphology, Cellular Viability, Osteogenic Differentiation, and mRNA Expression within Stem Cell Spheroids.

Background and Objectives: Tacrolimus is a macrolide lactone compound derived from the bacterium Streptomyces tsukubensis, widely known as an immunosuppressant. In basic research, the effects of tacrolimus on osteogenic differentiation have been tested using mesenchymal stem cells. In this study, tacrolimus's effects on the cellular survival and osteogenic differentiation of stem cell spheroids were investigated. Materials and Methods: Concave microwells were used to form stem cell spheroids in the presence of tacrolimus at final concentrations of 0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL. A microscope was used to test cellular vitality qualitatively, and an assay kit based on water-soluble tetrazolium salt was used to measure cellular viability quantitatively. Alkaline phosphatase activity and an anthraquinone dye test for measuring calcium deposits were used to assess osteogenic differentiation. To assess the expression of osteogenic differentiation, a quantitative polymerase chain reaction, Western blot, and RNA sequencing were performed. Results: Spheroids across all concentrations maintained a relatively uniform and spherical shape. Cell viability assay indicated that tacrolimus, up to a concentration of 100 µg/mL, did not significantly impair cell viability within spheroids cultured in osteogenic media. The increase in calcium deposition, particularly at lower concentrations of tacrolimus, points toward an enhancement in osteogenic differentiation. There was an increase in COL1A1 expression across all tacrolimus concentrations, as evidenced by the elevated mean and median values, which may indicate enhanced osteogenic activity. Conclusions: This study showed that tacrolimus does not significantly impact the viability of stem cell spheroids in osteogenic media, even at high concentrations. It also suggests that tacrolimus may enhance osteogenic differentiation, as indicated by increased calcium deposition and COL1A1 expression. These findings advance our understanding of tacrolimus's potential roles in tissue repair, regeneration, and stem cell-based therapeutic applications.

Long-term skeletodental changes with early and late treatment using modified C-palatal plates in hyperdivergent Class II adolescents.

OBJECTIVES: To compare skeletodental changes between early and late treatment groups using modified C-palatal plates (MCPP) and long-term retention outcomes in hyperdivergent Class II adolescents. MATERIALS AND METHODS: Seventy-one hyperdivergent Class II patients were divided into four groups according to treatment modality and treatment timing: group 1, early treatment with MCPP (n = 16; 9.9 ± 0.9 years); group 2, late treatment with MCPP (n = 19; 12.3 ± 0.8 years); group 3, early treatment with headgear (HG; n = 18; 9.6 ± 0.8 years); and group 4, late treatment with HG (n = 18; 12.1 ± 1.2 years). Lateral cephalograms were taken and skeletal and dental variables were measured. For statistical analysis, paired t-tests, independent t-tests, and multiple regression were performed. RESULTS: The early MCPP group showed a more significant decrease in mandibular plane angle than the late MCPP group did, and vertical control was more efficient in the early group than in the late group. In the MCPP groups, both FMA and SN-GoGn were increased with late treatment but decreased with early treatment, and the difference was statistically significant (P < .01). The early-treatment MCPP group had a significant decrease in SN-GoGn of 0.6° compared with an increase of 1.7° in the early treatment HG group (P < .01). Posttreatment stability of both the early and late MCPP groups was maintained in long-term retention. CONCLUSIONS: Early MCPP showed more significant vertical control than late MCPP. However, there was no difference in long-term stability between early and late groups.

Accuracy of posteroanterior cephalogram landmarks and measurements identification using a cascaded convolutional neural network algorithm: A multicenter study.

Objective: : To quantify the effects of midline-related landmark identification on midline deviation measurements in posteroanterior (PA) cephalograms using a cascaded convolutional neural network (CNN). Methods: : A total of 2,903 PA cephalogram images obtained from 9 university hospitals were divided into training, internal validation, and test sets (n = 2,150, 376, and 377). As the gold standard, 2 orthodontic professors marked the bilateral landmarks, including the frontozygomatic suture point and latero-orbitale (LO), and the midline landmarks, including the crista galli, anterior nasal spine (ANS), upper dental midpoint (UDM), lower dental midpoint (LDM), and menton (Me). For the test, Examiner-1 and Examiner-2 (3-year and 1-year orthodontic residents) and the Cascaded-CNN models marked the landmarks. After point-to-point errors of landmark identification, the successful detection rate (SDR) and distance and direction of the midline landmark deviation from the midsagittal line (ANS-mid, UDM-mid, LDM-mid, and Me-mid) were measured, and statistical analysis was performed. Results: : The cascaded-CNN algorithm showed a clinically acceptable level of point-to-point error (1.26 mm vs. 1.57 mm in Examiner-1 and 1.75 mm in Examiner-2). The average SDR within the 2 mm range was 83.2%, with high accuracy at the LO (right, 96.9%; left, 97.1%), and UDM (96.9%). The absolute measurement errors were less than 1 mm for ANS-mid, UDM-mid, and LDM-mid compared with the gold standard. Conclusions: : The cascaded-CNN model may be considered an effective tool for the auto-identification of midline landmarks and quantification of midline deviation in PA cephalograms of adult patients, regardless of variations in the image acquisition method.

Precautions and possibilities in orthodontic treatment of periodontally compromised patients: Current recommendations.

OBJECTIVE: Orthodontic treatment plays a crucial role in achieving optimal dental esthetics and functional occlusion. However, when periodontally compromised patients are involved, additional precautions and considerations are critical. This article aims to provide up-to-date recommendations for the orthodontic treatment of periodontally compromised patients. CLINICAL CONSIDERATIONS: Comprehensive diagnosis of the patient's periodontal status, inherent malocclusion, and secondary malocclusion resulting from periodontal disease are essential for achieving optimal esthetics and functional occlusion. This can be facilitated through the use of a simplified checklist. Prior to orthodontic treatment, pre-existing periodontal diseases should be managed. Light and controlled forces should be used to minimize the risk of adverse effects on the periodontium, and any potential traumatic occlusion during tooth movement should be minimized. Furthermore, careful anchorage management is required, and proper application of temporary anchorage devices can significantly expand the scope of orthodontic treatment. Finally, treatment results are maintained by ongoing supportive periodontal therapy even during the retention period. CONCLUSIONS: This article presents clinical cases demonstrating the importance of accurate diagnosis in orthodontics and periodontics and the positive impact of orthodontic treatment on patients with pre-existing periodontal diseases. CLINICAL SIGNIFICANCE: An up-to-date orthodontic treatment protocol for periodontally compromised patients is presented.

Comparative Efficacy of Traditional Corticotomy and Flapless Piezotomy in Facilitating Orthodontic Tooth Movement: A Systematic Review and Meta-Analysis.

Background and Objectives: This study aimed to perform a meta-analysis comparing the effects of corticotomy and flapless piezocision on accelerated tooth movement. Materials and Methods: A comprehensive search using a combination of controlled vocabulary (MeSH) and free-text terms was undertaken by two reviewers to identify published systematic reviews. Three major electronic databases (Medline via PubMed, Cochrane Database, and Embase) were searched up to 2 June 2023. Results: The results of the meta-analysis showed that the pooled standardized mean difference values of accumulative movement distances for flapless piezocision were 1.43 (95% CI, 0.38 to 2.48; p < 0.01), 1.09 (95% CI, -0.08 to 2.26; p = 0.07), and 0.73 (95% CI, -0.58 to 4.02; p = 0.14). The results of the meta-analysis demonstrated that the pooled SMD values of accumulative movement distances for the corticotomy were 2.76 (95% CI, 0.18 to 5.34; p = 0.04), 1.43 (95% CI, -1.10 to 3.96; p = 0.27), and 4.78 (95% CI, -4.54 to 14.10; p = 0.32). Although the test for overall effectiveness was significant for piezocision and corticotomy, there were no significant differences between piezocision and corticotomy. Conclusions: The study determined that both conventional corticotomy and flapless piezosurgery are effective as adjuncts to orthodontic treatment. Moreover, no significant difference was observed in the short-term effectiveness of canine retraction acceleration between conventional corticotomy and flapless piezocision. While piezocision may be a favorable option for orthodontic treatment, corticotomy can be considered in cases requiring additional procedures such as bone grafting.

Enterovibrio paralichthyis sp. nov., isolated from the gut of an olive flounder Paralichthys olivaceus.

A Gram-stain-negative, facultatively anaerobic, flagellated and coccoid, ovoid or rod-shaped bacterial strain, NIFS-20-8T, was isolated from the intestine of an olive flounder (Paralichthys olivaceus) from the East Sea, Republic of Korea. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain NIFS-20-8T fell within the clade comprising the type strains of Enterovibrio species. Strain NIFS-20-8T exhibited 16S rRNA gene sequence similarities of 97.2 and 97.1 % to the type strains of Enterovibrio nigricans and Enterovibrio norvegicus, respectively, and of 96.6-97.0 % to the type strains of the other Enterovibrio species. The average nucleotide identity and digital DNA-DNA hybridization values between the genomic sequence of strain NIFS-20-8T and those of the type strains of four Enterovibrio species were 73.8-75.0 and 19.8-21.1 %, respectively. The DNA G+C content of strain NIFS-20-8T from genomic sequence data was 50.55 mol%. Strain NIFS-20-8T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C18 : 1 ω7c as the major fatty acids. The major polar lipids detected in stain NIFS-20-8T were phosphatidylethanolamine and phosphatidylglycerol. Distinguishing phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain NIFS-20-8T is separated from recognized Enterovibrio species. On the basis of the data presented here, strain NIFS-20-8T is considered to represent a novel species of the genus Enterovibrio, for which the name Enterovibrio paralichthyis sp. nov. is proposed. The type strain is NIFS-20-8T (= KCTC 82873T=NBRC 115237T).

Evaluation of the radiation response and regenerative effects of mesenchymal stem cell-conditioned medium in an intestinal organoid system.

Intestinal organoids have recently emerged as an in vitro model relevant to the gut system owing to their recapitulation of the native intestinal epithelium with crypt-villus architecture. However, it is unclear whether intestinal organoids reflect the physiology of the in vivo stress response. Here, we systemically investigated the radiation response in organoids and animal models using mesenchymal stem cell-conditioned medium (MSC-CM), which contains secreted paracrine factors. Irradiated organoids exhibited sequential induction of viability loss and regrowth after irradiation (within 12 days), similar to the response of the native intestinal epithelium. Notably, treatment with MSC-CM facilitated the reproliferation of intestinal stem cells (ISCs) and restoration of damaged crypt-villus structures in both models. Furthermore, Wnt/Notch signaling pathways were commonly upregulated by MSC-CM, but not radiation, and pharmacologically selective inhibition of Wnt or Notch signaling attenuated the enhanced recovery of irradiated organoids, with increases in ISCs, following MSC-CM treatment. Interestingly, the expression of Wnt4, Wnt7a, and active ß-catenin was increased, but not notch family members, in MSC-CM-treated organoid after irradiation. Treatment of recombinant mouse Wnt4 and Wnt7a after irradiation improved to some extent intestinal epithelial regeneration both in vitro and in vivo. Overall, these results suggested that intestinal organoids recapitulated the physiological stress response of the intestinal epithelium in vivo. Thus, our findings provided important insights into the physiology of intestinal organoids and may contribute to the development of strategies to enhance the functional maturation of engineered organoids.

Cone-beam computed tomography analysis of transverse dental compensation in patients with skeletal Class III malocclusion and facial asymmetry.

OBJECTIVE: The purpose of this study was to analyze the transverse dental compensation in reference to the maxillary and mandibular basal bones using cone-beam computed tomography (CBCT) and evaluate the correlations between transverse dental compensation and skeletal asymmetry variables in patients with skeletal Class III malocclusion and facial asymmetry. METHODS: Thirty patients with skeletal Class I (control group; 15 men, 15 women) and 30 patients with skeletal Class III with menton deviation (asymmetry group; 16 men, 14 women) were included. Skeletal and dental measurements were acquired from reconstructed CBCT images using OnDemand3D 1.0 software. All measurements were compared between groups and between the deviated and nondeviated sides of the asymmetry group. Correlation coefficients for the association between skeletal and dental measurements were calculated. RESULTS: Differences in the ramus inclination (p < 0.001), maxillary canine and first molar inclinations (p < 0.001), and distances from the canine and first molar cusp tips to the midmaxillary or midmandibular planes (p < 0.01) between the right and left sides were significantly greater in the asymmetry group than in the control group. In the asymmetry group, the ramus inclination difference (p < 0.05) and mandibular canting (p < 0.05) were correlated with the amount of menton deviation. In addition, dental measurements were positively correlated with the amount of menton deviation (p < 0.05). CONCLUSIONS: Transverse dental compensation was correlated with the maxillary and mandibular asymmetry patterns. These results would be helpful in understanding the pattern of transverse dental compensation and planning surgical procedure for patients with skeletal Class III malocclusion and facial asymmetry.

Long-Term Culture of Intestinal Organoids.

The in vitro long-term expansion of primary intestinal epithelial cells has been hampered by the inability to maintain an immature stem cell population. Recent technical advances have led to the development of a novel in vitro culture system that can sustain intestinal stem cells (ISCs) using growth factors that mimic the intestinal microenvironment in combination with a three-dimensional (3D) culture. The resulting intestinal organoids display a crypt-villus architecture that recapitulates the native intestinal epithelium. Here, we describe our method for the long-term culture of intestinal epithelial organoids via consistent passaging using a gentle cell dissociation reagent to easily break the organoid into smaller pieces. The long-term cryopreservation and defining characteristics of these intestinal organoids also make this work relevant for the advancement of epithelial organoid-based therapeutic technologies by allowing the production of large numbers of cells for use in clinical applications.

Post-irradiation promotes susceptibility to reprogramming to pluripotent state in human fibroblasts.

Ionizing radiation causes not only targeted effects in cells that have been directly irradiated but also non-targeted effects in several cell generations after initial exposure. Recent studies suggest that radiation can enrich for a population of stem cells, derived from differentiated cells, through cellular reprogramming. Here, we elucidate the effect of irradiation on reprogramming, subjected to two different responses, using an induced pluripotent stem cell (iPSC) model. iPSCs were generated from non-irradiated cells, directly-irradiated cells, or cells subsequently generated after initial radiation exposure. We found that direct irradiation negatively affected iPSC induction in a dose-dependent manner. However, in the post-irradiated group, after five subsequent generations, cells became increasingly sensitive to the induction of reprogramming compared to that in non-irradiated cells as observed by an increased number of Tra1-81-stained colonies as well as enhanced alkaline phosphatase and Oct4 promoter activity. Comparative analysis, based on reducing the number of defined factors utilized for reprogramming, also revealed enhanced efficiency of iPSC generation in post-irradiated cells. Furthermore, the phenotypic acquisition of characteristics of pluripotent stem cells was observed in all resulting iPSC lines, as shown by morphology, the expression of pluripotent markers, DNA methylation patterns of pluripotency genes, a normal diploid karyotype, and teratoma formation. Overall, these results suggested that reprogramming capability might be differentially modulated by altered radiation-induced responses. Our findings provide that susceptibility to reprogramming in somatic cells might be improved by the delayed effects of non-targeted response, and contribute to a better understanding of the biological effects of radiation exposure.

Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.

AIM: To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells. METHODS: Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging. RESULTS: We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging. CONCLUSION: The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

Dual matrilineal geographic distribution of Korean type 2 diabetes mellitus-associated -11,377 G adiponectin allele.

The present study was performed to identify the susceptible single nucleotide polymorphisms (SNPs) for the prediction of Korean type 2 diabetes mellitus (T2DM) and to clarify the matrilineal origin of Korean T2DM­specific SNPs. Fourteen SNPs from the adiponectin (ADIPOQ), hepatocyte nuclear factor 4α, phosphoenolpyruvate carboxykinase 1 and glucokinase genes in the Korean population were analyzed. Only one SNP, ­11,377 C/G on the ADIPOQ gene, was finally determined to be responsible for the incidence of Korean T2DM (P=0.028). The G­T­T­A haplotype at positions ­11,377, +45, +276 and +349 on the ADIPOQ gene was also associated with a high incidence of Korean T2DM (P=0.023). In addition, the susceptibility of Korean individuals to T2DM appears to be affected by their matrilineal origin. Of note, the group of Southern origin, consisting of mitochondrial DNA macrohaplogroups F and R, was predisposed to T2DM, whereas the group of Northern origin, consisting of haplogroups A and Y, was resistant to T2DM. This implied that the differential genetics between the two groups, which were formed from the initial peopling of the proto­Korean population via Southern and Northern routes to the present time, may explain their differing susceptibility to T2DM. In conclusion, from Southern Asia Northward, a matrilineal origin of Korean individuals appears to be responsible for the prevalence of Korean T2DM caused by the ­11,377 G allele.

Evaluation of the relationship between upper incisor exposure and cephalometric variables in Korean young adults.

OBJECTIVE: The purpose of this study was to classify Korean young adults into 3 groups on the basis of upper incisor exposure rates (UIERs) and to compare the skeletal, dental, and soft tissue variables. METHODS: Samples were obtained from 127 students at the College of Dentistry, Wonkwang University in South Korea. Facial photographs of frontal posed smiles and lateral cephalograms of the subjects were taken. The subjects were divided into 3 groups on the basis of UIERs and 20 measurements were compared among the 3 groups. The correlations between the variables were determined. RESULTS: Male and female subjects showed significant differences in the group distribution. Male subjects showed higher frequencies of low smiles, and female subjects showed higher frequencies of high smiles. The vertical height of the anterior alveolar process of the maxilla directly correlated with the UIER. However, the UIER showed no significant correlation with the vertical height of the anterior basal bone or the inclination of the upper incisor axis. In female subjects, the upper central incisor clinical crown length showed an inverse correlation with the UIER. However, this variable showed no significant correlation with the UIER in male subjects. CONCLUSIONS: The UIER was directly correlated with the levator muscle activity of the upper lip and inversely correlated with the upper lip thickness, yet there was no correlation between the UIER and upper lip length at rest.

Rapid spontaneous reduction of a huge intracerebral hematoma.

Rapid reduction of a large acute subdural hematoma has been frequently reported. In my knowledge, however, it was rarely reported that rapid spontaneous reduction occurred in large volume of spontaneous intracerebral hematoma (sICH). We describe a patient with a rapid spontaneous decrease in the volume of a large hematoma. A 73-year-old man presented semi-comatose mentality. Initial brain computed tomography (CT) revealed the huge sICH. An emergency operation was planned, but was not performed due to the refusal of patient's family. Therefore, we decided to treat with conservative therapy. However, follow-up brain CT 16 hours after initial scan showed a remarkable reduction of previous sICH. The mechanism involving the spontaneous rapid decrease of the hematoma is presumed to occur through redistribution in brain atrophy, compression effect from the increased intracranial pressure and dilution through a wash out by the cerebrospinal fluid.

Single incision percutaneous pedicle screw fixation for transforaminal lumbar interbody fusion.

OBJECTIVE: Percutaneous pedicle screw fixation (PPSF) offers some of the advantages of minimally invasive surgery. However, an additional skin incision is necessary in order to gain these advantages. The author recently performed a modified PPSF through a single skin incision and it yielded good results. Here, we report the single incision percutaneous pedicle screw fixation (SIPPSF). METHODS: The medical records of 51 patients who underwent single segment transforaminal lumbar interbody fusion (TLIF) with pedicle screw fixation (PSF) between December 2009 and December 2011 were reviewed. The patients were divided into 3 groups according to the PSF technique used; conventional group (Group C), SIPPSF (Group S) and PPSF through multiple skin incisions (Group M). Operation time, estimated blood loss (EBL) and pain intensity before and after the operation using a numeric rating scale (NRS) were evaluated. RESULTS: The number of patients in Group S, Group C and Group M was 25, 17 and 9, respectively. The NRS demonstrated a reduction in pain intensity in all the patients, and there were no differences in NRS scoring between the three groups. Group S showed a reduction in the EBL compared to Group C (p=0.001) and a reduction in the operation time compared to Group M(p=0.003). CONCLUSION: The author supposes that the type of skin incision does not correlate significantly with the clinical results of TLIF with PPSF. However, it can be considered that SIPPSF offers the advantage of avoiding an additional paramedian skin incision.