Meniscus fibrochondrocytes (MFCs) may be the optimal cell source to repair non-healing meniscus injuries using tissue engineering strategies. In this study, we investigated the effects of mitotic divisions and oxygen tension on the plasticity of adult human MFCs. Our assessment techniques included gene expression, biochemical, histological, and immunofluorescence assays. MFCs were expanded in monolayer culture with combined growth factors TGFβ1 and FGF-2 (T1F2) under normoxia (21% O₂). Trilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation was performed under both normoxic (21% O₂) and hypoxic (3% O₂) conditions. The data demonstrated that MFCs with a mean total population doubling of 10 can undergo adipogenesis and chondrogenesis. This capability was enhanced under hypoxic conditions. The MFCs did not undergo osteogenesis. In conclusion, our findings suggest that extensively expanded human MFCs have the capacity to generate tissues with the functional matrix characteristics of avascular meniscus. To this end, expanded MFCs may be an ideal cell source for engineering functional constructs for the replacement or repair of avascular meniscus.

Background: The glycolytic enzyme, α-Enolase (ENO1), catalyzes the production of phosphoenolpyruvate from 2-phosphoglycerate, thereby enhancing glycolysis and contributing to tumor progression. In the present study, we aimed to determine the role of ENO1 in skin cutaneous melanoma (SKCM) and the potential underlying mechanism. Methods: The Sangerbox database was used to analyze the mRNA expression of ENO1 in SKCM. Western blotting was used to assess the levels of ENO1, c-Myc, β-catenin, MMP-9, PGAM1, and MMP-13 in SKCM-derived cell lines or tumor tissues from patients with SKCM. The pCMV-SPORT6-ENO1 and pET-28a-ENO1siRNA plasmids were used to overexpress and knockdown ENO1 in SKCM cells, respectively. To determine the function of ENO1 in the malignant behavior of SKCM cells, we performed a wound-healing assay, cell counting kit 8 assay, and transwell chamber analyses. The production of pyruvate and lactic acid in tumor cells was evaluated using their respective kits. Results: Compared with non-tumor tissues, ENO1 was found to be overexpressed in SKCM tissues. In SKCM cells, ENO1 overexpression promoted invasion, migration, and proliferation of tumor cells; increased pyruvate and lactate production; and increased β-catenin, MMP-9, MMP-13, and c-Myc levels. The opposite effects were observed in SKCM cells silenced for ENO1. Conclusions: These results indicate that ENO1 is involved in SKCM progression by enhancing the invasion and proliferation of tumor cells. In addition, ENO1 might have an important function in tumor cell glycolysis. Therefore, ENO1 represents a potential therapeutic target for treatment of SKCM.

Objective: To investigate hypoxia and TGF-β3 effects on inner meniscus-like extracellular matrix (ECM) formation and related gene expression by meniscus fibrochondrocytes (MFCs). Design: Aggregates of human MFCs were cultured for 3 weeks under hypoxia (3% O ₂ ) or normoxia (atmospheric O ₂ ) with or without TGF-β3 supplementation (10 ng/mL). Inner meniscus-like ECM formation was assessed by biochemistry, histology, and immunofluorescence. mRNA expression of ECM-related genes, TGF-β isoforms 1–3, and hypoxia-inducible factor-1 (HIF-1) targets were assessed by quantitative real-time polymerase chain reaction. Results: Hypoxia and TGF-β3 supplementation synergistically induced inner meniscus-like ECM formation at the protein level with similar effects on ECM-related gene expression. Hypoxia alone did not induce an inner meniscus-like ECM-forming response nor upregulate mRNA of TGF-β isoforms. Expression of HIF-1α and HIF-1 target genes suggested that HIF-1 was a possible contributor to the observed synergistic interactions of hypoxia and TGF-β3 supplementation. Conclusion: Hypoxia and TGF-β3 supplementation synergistically induced inner meniscus ECM formation by adult human MFCs. Hypoxia alone is insufficient to induce an inner meniscus ECM-forming response in this culture model. Impact Statement: The interactions of hypoxia and TGF-β3 in aggregates of human meniscus fibrochondrocytes are synergistic in nature, suggesting combinatorial strategies using these factors are promising for tissue engineering the inner meniscus regions. Hypoxia alone in the absence of TGF-β supplementation may be insufficient to initiate an inner meniscus-like extracellular matrix-forming response in this model.

MicroRNAs (miRNAs) have been identified as crucial modulators of gene expression and to play a role in palatogenesis. The aim of this study was to explore the potential role and regulatory mechanisms of miRNAs during palatogenesis. RNA-sequencing was performed to compare the RNA expression profiles of mouse embryonic palatal shelf (MEPS) tissue between an all-trans retinoic acid (ATRA)-induced group and control group, followed by reverse transcription–quantitative polymerase chain reaction for validation, demonstrating upregulated expression of miRNA-470-5p and downregulated expression of Fgfr1 in the ATRA-induced group. The specific binding sites of miRNA-470-5p that potentially govern Fgfr1 expression were predicted by miRanda and TargetScan. The relationship between miRNA-470-5p and Fgfr1 was validated in HEK293T cells by luciferase reporter assays, confirming that miR-470-5p acts directly on the Fgfr1 3′-untranslated region. Fgfr1 mRNA and FGFR1 protein levels were markedly downregulated in MEPS epithelial cells over-expressing miRNA-470-5p. Functional experiments in vitro with CCK-8, cell colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) staining assays revealed that upregulated miRNA-470-5p expression could inhibit the epithelial-mesenchymal transition (EMT) of MEPS epithelial cells by targeting Fgfr1. These findings provide a new molecular mechanism of cleft palate formation, which can inform the development of new treatment and/or prevention targets.