EEF1B2 regulates bone marrow-derived mesenchymal stem cells bone-fat balance via Wnt/ß-catenin signaling.

The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

Carboxypeptidase M modulates BMSCs osteogenesis-adipogenesis via the MAPK/ERK pathway: An integrated single-cell and bulk transcriptomic study.

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP.

Endogenus chondrocytes immobilized by G-CSF in nanoporous gels enable repair of critical-size osteochondral defects.

Injured articular cartilage is a leading cause for osteoarthritis. We recently discovered that endogenous stem/progenitor cells not only reside in the superficial zone of mouse articular cartilage, but also regenerated heterotopic bone and cartilage in vivo. However, whether critical-size osteochondral defects can be repaired by pure induced chemotatic cell homing of these endogenous stem/progenitor cells remains elusive. Here, we first found that cells in the superficial zone of articular cartilage surrounding surgically created 3 × 1 mm defects in explant culture of adult goat and rabbit knee joints migrated into defect-filled fibrin/hylaro1nate gel, and this migration was significantly more robust upon delivery of exogenous granulocyte-colony stimulating factor (G-CSF). Remarkably, G-CSF-recruited chondrogenic progenitor cells (CPCs) showed significantly stronger migration ability than donor-matched chondrocytes and osteoblasts. G-CSF-recruited CPCs robustly differentiated into chondrocytes, modestly into osteoblasts, and barely into adipocytes. In vivo, critical-size osteochondral defects were repaired by G-CSF-recruited endogenous cells postoperatively at 6 and 12 weeks in comparison to poor healing by gel-only group or defect-only group. ICRS and O'Driscoll scores of articular cartilage were significantly higher for both 6- and 12-week G-CSF samples than corresponding gel-only and defect-only groups. Thus, endogenous stem/progenitor cells may be activated by G-CSF, a Food and Drug Administration (FDA)-cleared bone-marrow stimulating factor, to repair osteochondral defects.

Identifying Lymph Nodes and Their Statuses from Pretreatment Computer Tomography Images of Patients with Head and Neck Cancer Using a Clinical-Data-Driven Deep Learning Algorithm.

BACKGROUND: Head and neck cancer is highly prevalent in Taiwan. Its treatment mainly relies on clinical staging, usually diagnosed from images. A major part of the diagnosis is whether lymph nodes are involved in the tumor. We present an algorithm for analyzing clinical images that integrates a deep learning model with image processing and attempt to analyze the features it uses to classify lymph nodes. METHODS: We retrospectively collected pretreatment computed tomography images and surgery pathological reports for 271 patients diagnosed with, and subsequently treated for, naïve oral cavity, oropharynx, hypopharynx, and larynx cancer between 2008 and 2018. We chose a 3D UNet model trained for semantic segmentation, which was evaluated for inference in a test dataset of 29 patients. RESULTS: We annotated 2527 lymph nodes. The detection rate of all lymph nodes was 80%, and Dice score was 0.71. The model has a better detection rate at larger lymph nodes. For those identified lymph nodes, we found a trend where the shorter the short axis, the more negative the lymph nodes. This is consistent with clinical observations. CONCLUSIONS: The model showed a convincible lymph node detection on clinical images. We will evaluate and further improve the model in collaboration with clinical physicians.

Temporal transcriptome features identify early skeletal commitment during human epiphysis development at single-cell resolution.

Human epiphyseal development has been mainly investigated through radiological and histological approaches, uncovering few details of cellular temporal genetic alternations. Using single-cell RNA sequencing, we investigated the dynamic transcriptome changes during post-conception weeks (PCWs) 15-25 of human distal femoral epiphysis cells. We find epiphyseal cells contain multiple subtypes distinguished by specific markers, gene signatures, Gene Ontology (GO) enrichment analysis, and gene set variation analysis (GSVA). We identify the populations committed to cartilage or ossification at this time, although the secondary ossification centers (SOCs) have not formed. We describe the temporal alternation in transcriptional expression utilizing trajectories, transcriptional regulatory networks, and intercellular communication analyses. Moreover, we find the emergence of the ossification-committed population is correlated with the COL2A1-(ITGA2/11+ITGB1) signaling. NOTCH signaling may contribute to the formation of cartilage canals and ossification via NOTCH signaling. Our findings will advance the understanding of single-cell genetic changes underlying fetal epiphysis development.

Single-cell transcriptome analysis reveals aberrant stromal cells and heterogeneous endothelial cells in alcohol-induced osteonecrosis of the femoral head.

Alcohol-induced osteonecrosis of the femoral head (ONFH) is a disabling disease with a high incidence and elusive pathogenesis. Here, we used single-cell RNA sequencing to explore the transcriptomic landscape of mid- and advanced-stage alcohol-induced ONFH. Cells derived from age-matched hip osteoarthritis and femoral neck fracture samples were used as control. Our bioinformatics analysis revealed the disorder of osteogenic-adipogenic differentiation of stromal cells in ONFH and altered regulons such as MEF2C and JUND. In addition, we reported that one of the endothelial cell clusters with ACKR1 expression exhibited strong chemotaxis and a weak angiogenic ability and expanded with disease progression. Furthermore, ligand-receptor-based cell-cell interaction analysis indicated that ACKR1+ endothelial cells might specifically communicate with stromal cells through the VISFATIN and SELE pathways, thus influencing stromal cell differentiation in ONFH. Overall, our data revealed single cell transcriptome characteristics in alcohol-induced ONFH, which may contribute to the further investigation of ONFH pathogenesis.

Alkaline activation of endogenous latent TGFß1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFß1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFß1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFß1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Combining bulk and single-cell RNA-sequencing data to reveal gene expression pattern of chondrocytes in the osteoarthritic knee.

Osteoarthritis (OA) occurs mostly in the knees, hips, finger interphalangeal joints, and spinal facet joints, and is characterized by cartilage degeneration. The existing bulk RNA sequencing (bulk RNA-seq) and single-cell sequencing (scRNA-seq) data for chondrocytes in the osteoarthritic knee joint provide the expression profiles of entire cell populations and individual cells, respectively. Here, we aimed to analyze these two types of sequencing data in order to obtain a more comprehensive understanding of OA. We compared the analysis results of bulk RNA-seq and scRNA-seq from the dataset GSE114007 and the dataset GSE104782, respectively, and identified the differentially expressed genes (DEGs). Then, we tried to find the key The transcription factor is a more fomal term (TFs) and long non-coding RNA (lncRNA) regulation. We highlighted 271 genes that were simultaneously suggested by these two types of data and provided their possible expression pattern in OA. Among the 271 genes, we identified 14 TFs, and TWIST2, MYBL2, RELA, JUN, KLF4, and PTTG1 could be the key TFs for the 271 genes. We also found that 8 lncRNAs among the 271 genes and the lncRNA regulation between CYTOR and NRP1 could contribute to the pain and vascularization of cartilage in the osteoarthritic knee. In short, our research combined the analysis results of bulk RNA-seq and scRNA-seq data for OA chondrocytes, which will contribute to further elucidation of the molecular mechanisms of OA pathogenesis.[Figure: see text].

[Observation of dendrite osteocytes of mice at different developmental stages using Ploton silver staining and phalloidin staining].

OBJECTIVE: To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages. METHODS: The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone. RESULTS: In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 vs P28, P < 0.05; P21 vs P35, P < 0.05). In the humeral cortical bone of P15 mice, the morphology of the osteocytes and canalicular could be observed with Ploton silver staining, and the length of the regularly arranged canaliculi of the osteocytes increased significantly with age (P15 vs P21, P < 0.005; P15 vs P28, P < 0.0001; P15 vs P35, P < 0.0001). Phalloidin iFlour-488 staining was capable of visualizing the complete morphology of the osteocytes at P10 and P15; the osteocyte dendrites elongated progressively with age (P10 vs P15, P < 0.01) to form connections with the surrounding osteocytes. CONCLUSIONS: Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.

[An optimized method for embedding undecalcified mouse tibias in plastic blocks].

OBJECTIVE: To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. METHODS: Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO2 was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. RESULTS: We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO2 into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections (P < 0.05) without affecting bone formation analysis (P > 0.05). Heating the slides significantly lowered the detachment rate of the sections (P < 0.05) without affecting osteoblast analysis (P > 0.05). CONCLUSIONS: The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.

[Effect of polycaprolactone-ascobic acid scaffold in repairing articular cartilage defects in rabbits].

OBJECTIVE: To investigate the effect of polycaprolactone-ascobic acid (PCL-AA) scaffolds in promoting repair of articular cartilage defects in a rabbit model. METHODS: The cartilage defects (3.5 mm in diameter and 3.0 mm in depth) were created in the trochlear groove of the bilateral knees of eight 6-month-old male New Zealand white rabbits. The rabbit models were then randomized into 3 groups to receive implantation of PCL-AA scaffolds (group A, n=8), implantation of PCL scaffolds without AA (group B, n=5), or no treatment (group C, n=3). In groups A and B, the mixture of fibrin gel (10 µg) and thrombinogen (10 µg) was injected into the defects to fix the scaffolds during the surgery. Histological analyses and quantitative assessments of defect repair were conducted at 6 and 12 weeks after implantation of the scaffold. RESULTS: At 6 weeks after scaffold implantation, macroscopic observation showed better filling of the cartilage defects in group A than in group B, while no obvious defect repair was observed in group C. The rabbits in group A showed a significant improvement of the Wakitani score than those in group B (4.05∓1.11 vs 7.05∓0.98, P<0.05). HE staining revealed the presence of newly generated cells in and around the PCL-AA scaffolds without inflammatory cells. Safranin O staining showed a significantly greater ECM of the newly regenerated tissue in groups A and B than in group C (P<0.05), and the volume of the regenerated cartilage and cells was significantly greater in group A than in group B (P<0.05). Samples harvested at 12 weeks showed more hyalione-like cartilage formation than that at 6 weeks in group A. CONCLUSION: PCL-AA scaffolds have a good biocompatibility and promotes the healing of articular cartilage defects. Adding ascorbic acid into PCL scaffolds better promotes cartilage formation in terms of both quantity and quality of the regenerated tissues. PCL-AA scaffolds can serve as a promising biomaterial to promote the regeneration of articular cartilage using tissue engineering techniques.