Identification of Cuproptosis-related Gene Lipoyltransferase 1 as a Promising Biomarker in Oral Squamous Cell Carcinoma.

OBJECTIVE: To find efficient cuproptosis-related biomarkers to explore the oncogenesis and progression of oral squamous cell carcinoma (OSCC). METHODS: All the original data were downloaded from the Cancer Genome Atlas (TCGA) database. Univariate Cox analysis and Kaplan-Meier survival analysis were used to identify the gene related to survival. Tumor Immune Estimation Resource 2.0 (TIMER 2.0) was used to reveal the different expression of cuproptosis-related gene lipoyltransferase 1 (LIPT1) in various kinds of tumours. RESULTS: LIPT1, as a cuproptosis-related gene, was found to be differentially expressed in the OSCC group and the control group. It was also found to be related to the prognosis of OSCC. Pan cancer analysis showed LIPT1 was also involved in various kinds of tumours. CONCLUSION: All the results demonstrate that the cuproptosis-related gene LIPT1 is highly involved in the oncogenesis and progression of OSCC. These findings give new insight for further research into the cuproptosis-related biomarkers in OSCC.

A review of tamoxifen administration regimen optimization for Cre/loxp system in mouse bone study.

Gene knockout is a technique routinely used in basic experimental research, particularly in mouse skeletal and developmental studies. Tamoxifen-induced Cre/loxp system is known for its temporal and spatial precision and commonly utilized by researchers. However, tamoxifen has been shown its side effects on affecting the phenotype of mouse bone directly. This review aimed to optimize tamoxifen administration regimens including its dosage and duration, to identify an optimal induction strategy that minimizes potential side effects while maintaining recombination efficacy. This study will help researchers in designing gene knockout experiments in bone when using tamoxifen.

[Application of Animal Model Training in Preclinical Skills Teaching for Graduate Students of Dentistry].

Objective: To explore the potential application value of animal model training in improving the comprehensive clinical ability of postgraduate students of dentistry and to provide reference for new methods of preclinical skills teaching. Methods: A total of 40 postgraduate students of dentistry were assigned to two groups, an experimental group and a control group. The control group took the routine teaching course on root canal treatment for the right mandibular first molar, using a simulated model of human head. The experimental group also took a teaching course on root canal therapy for the right mandibular first molar, but an animal model was used for the group. After the course was completed, the instructor conducted comprehensive evaluation of the students' psychological quality, patient communication skills, diagnosis and treatment logic, speed of performing procedures, and treatment plan design. A questionnaire survey was conducted to examine the students' attitudes toward and evaluation of animal model training. Results: The scores for psychological quality (0.430±0.024 vs. 0.115±0.036), patient communication skills (0.878±0.065 vs. 0.115±0.036), diagnosis and treatment logic (0.630±0.066 vs. 0.372±0.033), speed of performing procedures (0.8975±0.019 vs. 0.055±0.080), and treatment plan design (0.539±0.036 vs. 0.396±0.017) of the experimental group were significantly higher than those of the control group ( P<0.0001). The total score of the experimental group (3.374±0.184) was significantly higher than that of the control group (1.053±0.082) and the difference was statistically significant ( P<0.001). 95% of the students in the control group and 100% of those in the experimental group were willing to participate in animal model training to improve their level of diagnosis and treatment skills for dental and endodontic diseases, showing no statistically significant difference ( χ 2=1.026, P=0.3112). In the experimental group, 30% of the students believed that their psychological qualities had been improved, 50% believed that their procedure skills had been improved, and 20% believed that animal model training had expanded the scope of their theoretical knowledge. Conclusion: Adding animal model training can improve dentistry graduate students' comprehensive abilities, including their psychological quality, patient communication skills, diagnosis and treatment logic, speed of performing procedures, and treatment plan design. In addition, it helps students familiarize themselves in advance with animal experimental operations for basic research, thus helping them acquire dual professional skills.

[Latest Research Findings on the Regulation of Bone Homeostasis by ALK3].

Bone remodeling, which is well orchestrated by osteogenesis of osteoblasts and osteoclastogenesis of osteoclasts, maintains the homeostasis of osteal development and metabolism under physiological conditions. Bone morphogenetic protein receptor type 1A, also known as activin receptor-like kinase 3 (ALK3), which exists on cytomembrane, is one of the key receptors of BMP factors, and is an important "gateway" that regulates the entrance of BMP signaling into cells in order to perform biological functions. The roles of BMP signaling in bone remodeling have been extensively studied. Many new discoveries have been reported in recent years through research based on transgenic mice models and focused on ALK3 as targets, shedding new light on the regulations of bone remodeling, cartilage and joint development, and the occurrence and treatment of bone-related diseases. Established understanding has been expanded, but new challenges on existing clinical application of BMPs also appeared. Hence, we reviewed recent studies on ALK3's involvement in bone formation and bone resorption, analyzed its mechanism of action in bone regulation, summarized the roles of ALK3 in the development of cartilage and temporomandibular joint, and reported the latest progress in treatment in preclinical studies, intending to provide references for subsequent studies and clinical applications in the future.

[Research Progress of Fibroblast Growth Factor 8's Role in the Regulation of Bone Development and Homeostasis].

The proper development and the homeostasis maintenance of bones are important prerequisites for the normal functioning of the human body. Bone developmental deformities or homeostasis disorders, such as Kashin-Beck disease, craniosynostosis, cleft palate and osteoarthritis, severely affect the life of patients, causing significant stress to the family and the society. Fibroblast growth factor 8 (FGF8) plays multiple functions through the course of the life of organisms. Abnormal expression of FGF8 may cause disorders of bone homeostasis and developmental abnormalities of bones. More and more studies have found that FGF8 may play an important role in bone development and may become a potential therapeutic target. Herein, we reviewed the role of FGF8 in a variety of skeletal abnormalities, intending to provide new perspectives for the prevention and treatment of related diseases in the future.

[Co-Culturing of Osteoblasts and Chondrocytes Upregulates HIF-1 Pathway of Chondrocytes via MAPK Signaling].

OBJECTIVE: To study the effect of co-culturing chondrocytes with osteoblasts on hypoxia-inducible factor (HIF)-1 pathway of chondrocytes and its mechanism. METHODS: Chondrocytes and osteoblasts were separately extracted from the knee joint and skull of newborn mice by trypsin digestion. The co-culturing system of osteoblasts and chondrocytes was constructed by using Transwell inserts to culture the osteoblasts and 6-well plate to culture the chondrocytes. We used qRT-PCR to examine changes in the mRNA expression of HIFs and its target gene pyruvate dehydrogenase kinase 1 ( PDK1) in chondrocytes co-cultured for 24 h. Western blot was used to analyze changes in the protein expression of HIFs and PDK1 and the changes in the activation of mitogen activated protein kinase (MAPK) signaling pathway after the cells were co-cultured for 48 h. Reactive oxygen species (ROS) staining was done to show the changes of ROS production in chondrocytes co-cultured for 48 h. RESULTS: The results of qRT-PCR and Western blot showed upregulated levels of HIF-1α gene and protein expression ( P<0.05) in the chondrocytes after they were co-cultured with osteoblasts. The gene and protein expression levels of PDK1 , the target gene of HIF-1, were also upregulated ( P<0.05). ROS staining showed that co-culturing of chondrocytes with osteoblasts decreased ROS production in chondrocytes. Western blot revealed that extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling of co-cultured chondrocytes were enhanced ( P<0.05). CONCLUSION: Co-culturing with osteoblasts enhanced the ERK1/2 and p38 signaling of chondrocytes and upregulated the HIF-1 pathway of chondrocytes.

[Research Progress in Glucose Metabolism of Chondrocytes].

Chondrocytes have a limited supply of glucose and oxygen for metabolism since articular cartilages are relatively avascular. We herein reviewed the characteristics of chondrocyte glucose metabolism and the new research progress in chondrocyte glucose metabolism in the osteoarthritis process. Current research has shown that chondrocytes obtain glucose from synovial fluids and subchondral bones, take in glucose via specific glucose transporters, and metabolize glucose mainly through glycolysis and mitochondrial respiration to produce adenosine triphosphate (ATP). Glucose metabolism in chondrocytes is distinctive because it relies much more on glycolysis rather than mitochondrial respiration for ATP production, and shows Warburg effect and Crabtree effect. In osteoarthritic chondrocytes, the glucose metabolism disorder is presented as further suppression of mitochondrial respiration, over-active or impaired glycolysis, and decreased total production of ATP. However, the significance of the glucose supply for chondrocytes from synovial fluids and subchondral bones remains undefined. There are still disputes in the understanding of the changes in glycolytic pathways in osteoarthritic chondrocytes. Therefore, future research is needed to explore the characteristics of glucose metabolism in normal and osteoarthritic chondrocytes in order to develop new diagnostic and therapeutic strategies for osteoarthritis.

[The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells].

OBJECTIVE: To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. METHODS: DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RESULTS: RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . CONCLUSION: RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

Osteogenesis, Osteoclastogenesis and their Crosstalk in Lipopolysaccharide-induced Periodontitis in Mice.

OBJECTIVE: To determine the crosstalk of osteogenesis and osteoclastogenesis of alveolar bone in lipopolysaccharide (LPS)-induced periodontitis in mice. METHODS: A representative periodontitis model was established by treating mice with LPS, and osteoblasts and osteoclasts were cultured. Osteoblasts and osteoclasts were cocultured to determine the effects of LPS on the crosstalk of osteogenesis and osteoclastogenesis. Quantitative polymerase chain reaction (qPCR) was performed to determine the expression of osteoclastogenesis makers underlying the potential mechanisms. RESULTS: The morphological and pathological changes in alveolar bone were observed in LPSinduced mice and LPS dose-dependently suppressed osteogenesis. The mRNA expression of cathepsin K, as a marker of osteoclasts, was accordingly downregulated in the coculture. The mRNA expression of osteoprotegerin was increased, while that of receptor activator of nuclear factor-κB ligand (RANKL) was decreased with an increased concentration of LPS. Moreover, the mRNA expression of toll-like receptor 4 (TLR4) was upregulated by LPS, whereas TLR4 knockout partially recovered osteoclast differentiation in the upper layer of the coculture. CONCLUSION: LPS dose-dependently suppressed osteogenesis but had a bidirectional effect on osteoclastogenesis. The combined effects of LPS on osteogenesis, osteoclastogenesis and their crosstalk via TLR4 account for alveolar bone loss in periodontitis.

[The Role of Connexins and Pannexins in the Cell Communications of Bone Cells].

Connexins and Pannexins play important roles in osteocytes and osteoblasts differentiation, intracellular signal transduction, maintenance of bone balance, and bone regeneration. This article reviews the progress and limitations of Connexins-mediated gap junctions and Pannexins mediated hemichannel in bone. Current research has shown that these molecules, in the form of gap junctions or separate hemichannels, deliver external stimuli to the skeletal system. However, little is known about the role of other cell types in bone development and homeostasis, such as pre-osteoblasts and bone marrow mesenchymal stem cells, in maintaining normality. In addition, at present, the most well-studied member of the Connexins family is Connexin43 (Cx43), while the roles and mechanisms of other members in bone development are still behind the veil. Gene-edited animal models provide basic information on the role of Connexins and Pannexins in the skeletal system, but the similarities and differences between Connexins and Pannexins remain to be discovered. Targeting a specific function of Connexins or Pannexins for bone stimulation and bone disease remains a challenge, with pharmacological selective overlap between channels, compensation of other subtypes, differences in methods for assessing channel function, and genetic changes associated with transgenic mouse models. Therefore, better tools and research pathways are needed to understand the role of these pathways in bone and cartilage. An essential task for future research will be to identify specific compounds that regulate Connexins or Pannexins subtypes to enable them to be used as pharmaceutical agents in the treatment of bone diseases, providing the possibility to develop new therapeutic strategies for improving bone health and treating diseases of the skeletal system.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency.

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption.