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Enhancer of zeste homolog 2 (EZH2) is an important member of the epigenetic regulatory factor polycomb group proteins (PcG) and is abnormally expressed in a wide variety of tumors, including osteosarcoma. Scientists consider EZH2 as an attractive target for the treatment of osteosarcoma and have found many potential EZH inhibitors, such as GlaxoSmithKline 343 (GSK343). It has been reported that GSK343 can be used as an inhibitor in different types of cancer. This study demonstrated that GSK343 not only induced apoptosis by increasing cleaved Casp-3 and poly ADP-ribose polymerase (PARP) expression, but also induced autophagic cell death by inhibiting p62 expression. Apoptosis and autophagic cell death induced by GSK343 were confirmed by the high expression of cleaved caspase-3, LC3-II and transmission electron microscopy. GSK343 inhibited the expression of EZH2 and c-Myc. Additionally, GSK343 inhibited the expression of FUSE binding protein 1 (FBP1), which was identified by its regulatory effects on c-Myc expression. Since c-Myc is a common target of EZH2 and FBP1, and GSK343 inhibited the expression of these proliferation-promoting proteins, a mutual regulatory mechanism between EZH2 and FBP1 was proposed. The knockdown of EZH2 suppressed the expression of FBP1; similarly, the knockdown of FBP1 suppressed the expression of EZH2. These results suggest the mutual regulatory association between EZH2 and FBP1. The knockdown of either EZH2 or FBP1 accelerated the sensitivity of osteosarcoma cells to GSK343. Based on these results, this study clarified that GSK343, an EZH2 inhibitor, may have potential for use in the treatment of osteosarcoma. The underlying mechanisms of the effects of GSK343 are partly mediated by its inhibitory activity against c-Myc and its regulators (EZH2 and FBP1). [ABSTRACT FROM AUTHOR]

A Large scale of full-thickness skin defects is lack of auto-grafts and which requires the engineered skin substitutes for repair and regeneration. One major obstacle in skin tissue engineering is to expand epidermal stem cells (ESCs) and develop functional substitutes. The other one is the scaffold of the ESCs. Here, we applied type I collagen-modified chitin membrane to form collagen-chitin biomimetic membrane (C-CBM), which has been proved to have a great biocompatibility and degraded totally when it was subcutaneously transplanted into rat skin. ESCs were cultured, and the resulting biofilm was used to cover full-thickness skin defects in nude mice. The transplantation of ESCs- collagen- chitn biomimetic membrane (ESCs-C-CBM) has achieved in situ skin regeneration. In nude mice, compared to controls with collagen-chitin biomimetic membrane (C-CBM) only, the ESCs-C-CBM group had significantly more dermatoglyphs on the skin wound 10 w after surgery, and the new skin was relatively thick, red and elastic. In vivo experiments showed obvious hair follicle cell proliferation in the full-thickness skin defect. Stem cell markers examination showed active ESCs in repair and regeneration of skin. The results indicate that the collagen-modified chitin membrane carry with ESCs has successfully regenerated the whole skin with all the skin appendages and function. [ABSTRACT FROM AUTHOR]

Background: Single-cell RNA sequencing (scRNA-seq) enables specific analysis of cell populations at single-cell resolution; however, there is still a lack of single-cell-level studies to characterize the dynamic and complex interactions between osteoporotic vertebral compression fractures (OVCFs) and Kümmell's disease (KD) in the osteoimmune microenvironment. In this study, we used scRNA-seq analysis to investigate the osteoimmune microenvironment and cellular composition in OVCFs and KD. Methods: ScRNA-seq was used to perform analysis of fractured vertebral bone tissues from one OVCF and one KD patients, and a total of 8,741 single cells were captured for single-cell transcriptomic analysis. The cellularity of human vertebral bone tissue was further analyzed using uniform manifold approximation and projection. Pseudo-time analysis and gene enrichment analysis revealed the biological function of cell fate and its counterparts. CellphoneDB was used to identify the interactions between bone cells and immune cells in the osteoimmune microenvironment of human vertebral bone tissue and their potential functions. Results: A cellular profile of the osteoimmune microenvironment of human vertebral bone tissue was established, including mesenchymal stem cells (MSCs), pericytes, myofibroblasts, fibroblasts, chondrocytes, endothelial cells (ECs), granulocytes, monocytes, T cells, B cells, plasma cells, mast cells, and early erythrocytes. MSCs play an immunoregulatory function and mediate osteogenic differentiation and cell proliferation. The differentiation trajectory of osteoclasts in human vertebral bone tissue was also revealed. In addition, ECs actively participate in inflammatory infiltration and coupling with bone cells. T and B cells actively participate in regulating bone homeostasis. Finally, by identifying the interaction of ligand-receptor pairs, we found that immune cells and osteoclasts have bidirectional regulatory characteristics, have the effects of regulating bone resorption by osteoclasts and promoting bone formation, and are essential for bone homeostasis. It is also highlighted that CD8-TEM cells and osteoclasts might crosstalk via CD160-TNFRSF14 ligand-receptor interaction. Conclusion: Our analysis reveals a differential landscape of molecular pathways, population composition, and cell-cell interactions during OVCF development into KD. OVCFs exhibit a higher osteogenic differentiation capacity, owing to abundant immune cells. Conversely, KD results in greater bone resorption than bone formation due to depletion of MSCs and a relatively suppressed immune system, and this immune imbalance eventually leads to vertebral avascular necrosis. The site of action between immune cells and osteoclasts is expected to be a new therapeutic target, and these results may accelerate mechanistic and functional studies of osteoimmune cell types and specific gene action in vertebral avascular necrosis and pathological bone loss diseases, paving the way for drug discovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor XF declared a shared affiliation with the author JX at the time of review. The authors declare that they were editorial board members of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Xu, Huang, Li, Dai, Wu, Wang, Yao, Luo, Liu, Yang, Feng, Miao, Xu and Ye.)

Background: Bone-tendon junction (BTJ) is a unique structure connecting tendon and bone through a fibrocartilage zone. Owing to its unique structure, the regeneration of BTJ remains a challenge. Here, we study the fibrochondrogenic differentiation of human tendon-derived stem/progenitor cells (TSPCs) both in vitro and in vivo ., Methods: TSPCs were isolated from human patellar tendon tissues and investigated for their multidifferentiation potential. TSPCs were cultured in chondrogenic medium with transforming growth factor beta 3 (TGF-β3) and BMP-2 in vitro  ​and examined for the expression of fibrochondrogenic marker genes by quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence. TSPCs pretreated were also seeded in collage II sponge and then transplanted in immunocompromised nude mice to examine if the fibrochondrogenic characteristics were conserved in vivo ., Results: We found that TSPCs were differentiated towards fibrochondrogenic lineage, accompanied by the expression of collagen I, collagen II, SRY-box transcription factor 9 (Sox 9), and tenascin C. Furthermore, after TSPCs were seeded in collagen II sponge and transplanted in immunocompromised nude mice, they expressed fibrochondrogenic genes, including proteoglycan, collagen I, and collagen II., Conclusion: Taken together, this study showed that TSPCs are capable of differentiating towards fibrocartilage-like cells, and the fibrochondrogenic characteristics were conserved even in vivo , and thus might have the potential application for fibrocartilage regeneration in BTJ repair., The Translational Potential of This Article: TSPCs are able to differentiate into fibrocartilage-like cells and thus might well be one potential cell source for fibrocartilage regeneration in a damaged BTJ repair., (© 2019 Published by Elsevier (Singapore) Pte Ltd on behalf of Chinese Speaking Orthopaedic Society.)

Objective: To explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor a (TNF-alpha) and serum deprivation., Methods: The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups: Vit C group (group A), TNF-alpha group (group B), and serum deprivation group (group C). Group A was reassigned to Al subgroup (basic medium), A2 subgroup (100 pg/mL Vit C), and A3 subgroup (200 pg/mL Vit C). Group B was reassigned to B0 subgroup (control group), Bi subgroup (100 ng/mL TNF-alpha), B2 subgroup (100 microg/mL Vit C+100 ng/mL TNF-alpha), and B3 subgroup (200 microg/mL Vit C+100 ng/mL TNF-alpha). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2% FBS+100 microg/mL Vit C), and C3 subgroup (2% FBS+200 microg/mL Vit C). After application of 100 pg/mL or 200 microg/mL Vit C for 24 hours, NP cells were stimulated by TNF-alpha and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type I, collagen type II, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. Results Group A: Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with Al subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type I, collagen type II, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B: TNF-alpha significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C: 2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05)., Conclusion: Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 microg/mL Vit C may delay the apoptosis induced by TNF-alpha and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.

Objective: To investigate the protective effect of mannitol therapy on the vital organs and explore the underlying mechanisms in New Zealand rabbits with severe burn injury., Methods: Twelve New Zealand rabbits with severe burn injury (30% of TBSA) were randomized to receive fluid resuscitation with saline (control) or mannitol therapy starting at 1 h after the injury. Serum and urine samples were collected before and at 1, 4, 8, 24, and 48 h after the injury for detection of TNF-α, IL-6, ALT, AST, GGT, CK, CK-MB, BUN and Cr levels using sandwich ELISA., Results: One hour after sever burn injury, the serum levels of TNF-α and IL-6 began to increase along with ALT, AST, GGT, CK, CK-MB, BUN and Cr levels. Compared with control group, the rabbits in mannitol group showed significantly higher 48 h urine excretion of TNF-α (145 ± 8 vs 78 ± 1 0 pg/ml, P<0.05) and IL-6 (93 ± 6 vs 40 ± 8 pg/ml, P<0.05) but with lowered serum levels of TNF-α (0.62 ± 0.02 vs 0.83 ± 0.02 pg/ml, P<0.05) and IL-6 (0.45 ± 0.03 vs 0.56 ± 0.03 pg/ml, P<0.05) as well as lowered serum ALT, AST, GGT, CK, CK-MB, BUN and Cr levels (P<0.05)., Conclusion: In rabbits with severe burn injury, mannitol therapy can decrease serum TNF-α and IL-6 levels early after the injury to ameliorate potential functional impairment of the heart, liver and kidneys.

The preparation of collagen sponges was studied in order to develop tissue engineering scaffolds. Collagen solutions with varying concentrations were obtained by condensing the initial collagen with polyethylene glycol (PEG) at 4 degrees C for different periods of time, and then were freeze-dried to make collagen scaffolds. The porous characteristics of the prepared scaffolds were characterized by use of different methods, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM) and tensile tests. All collagen sponges were shown to have similar interconnected porous structures but were found to have different pore size, porosity, water capacity and the mechanical property, depending on the concentration of collagen solutions. These findings indicate that the way of controlling the concentration of collagen solutions with PEG permits the freeze-drying fabrication of collagen sponges with varying porous features suitable for different tissue engineering purposes.

In search of the optimal preparation method for large-scale purification of human plasma fibronectin, we adopted affinity chromatography with gelatin and the Sepharose 4B activated with cyanogen bromide to purify fibronectin from type "C" plasma of healthy males, and scanned the best method under the conditions of different amount of plasma loading and different residence time in column. In a given column volume of gelatin, the absorbent was related with the plasma residence time in column and the total amount of plasma loaded. As a result, the optimal loading amount of plasma is 150 ml, and the residence time is 20 minutes. The preparation method, herein, has been proved to require small amount of plasma and yield large amount of fibronectin.