Temporal transcriptome highlights the involvement of cytokine/JAK/STAT3 signaling pathway in the osteoinduction of BMSCs.

BACKGROUND: Mesenchymal stem cells (MSCs)-based therapy offers an effective strategy for bone regeneration to solve the clinical orthopedic problems. However, the transcriptional regulation of multiple transitional stages of continuous osteogenesis from MSCs has not been fully characterized. METHODS: Bone marrow mesenchymal stem cells (BMSCs) stimulated with osteogenic induction media were utilized to construct the in vitro osteogenic differentiation model. BMSCs were harvested after induction for 0, 7, 14 and 21 days, respectively, to perform the mRNA-sequencing (mRNA-Seq). The transcription factor networks and common molecules during the osteogenesis were revealed by using the temporal transcriptome. Further verification was performed by the quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence and Western blotting. RESULTS: It showed that BMSCs could differentiate into osteogenic, and crucial regulator in the MAPK signaling pathway, the PPAR signaling pathway, the Toll-like receptor signaling and the Cytokine/JAK/STAT signaling pathway. PPI protein interaction analysis also suggested that three cytokines are involved in osteogenic differentiation as core genes, including leukemia inhibitory factor (LIF), interleukin-6 (IL6) and colony-stimulating factor 3 (CSF3). The osteogenic process was negatively affected by the inhibition of JAK/STAT3 signaling pathway. CONCLUSIONS: This work might provide new insights in the crucial features of the transcriptional regulation during the osteogenesis, as well as offer important clues about the activity and regulation of the relatively long-activated Cytokine/JAK/STAT3 signaling pathway in osteoinduction of BMSCs.

Cardioprotective effect of ultrasound-targeted destruction of Sirt3-loaded cationic microbubbles in a large animal model of pathological cardiac hypertrophy.

Pathological cardiac hypertrophy occurs in response to numerous increased afterload stimuli and precedes irreversible heart failure (HF). Therefore, therapies that ameliorate pathological cardiac hypertrophy are urgently required. Sirtuin 3 (Sirt3) is a main member of histone deacetylase class III and is a crucial anti-oxidative stress agent. Therapeutically enhancing the Sirt3 transfection efficiency in the heart would broaden the potential clinical application of Sirt3. Ultrasound-targeted microbubble destruction (UTMD) is a prospective, noninvasive, repeatable, and targeted gene delivery technique. In the present study, we explored the potential and safety of UTMD as a delivery tool for Sirt3 in hypertrophic heart tissues using adult male Bama miniature pigs. Pigs were subjected to ear vein delivery of human Sirt3 together with UTMD of cationic microbubbles (CMBs). Fluorescence imaging, western blotting, and quantitative real-time PCR revealed that the targeted destruction of ultrasonic CMBs in cardiac tissues greatly boosted Sirt3 delivery. Overexpression of Sirt3 ameliorated oxidative stress and partially improved the diastolic function and prevented the apoptosis and profibrotic response. Lastly, our data revealed that Sirt3 may regulate the potential transcription of catalase and MnSOD through Foxo3a. Combining the advantages of ultrasound CMBs with preclinical hypertrophy large animal models for gene delivery, we established a classical hypertrophy model as well as a strategy for the targeted delivery of genes to hypertrophic heart tissues. Since oxidative stress, fibrosis and apoptosis are indispensable in the evolution of cardiac hypertrophy and heart failure, our findings suggest that Sirt3 is a promising therapeutic option for these diseases. STATEMENT OF SIGNIFICANCE: Pathological cardiac hypertrophy is a central prepathology of heart failure and is seen to eventually precede it. Feasible targets that may prevent or reverse disease progression are scarce and urgently needed. In this study, we developed surface-filled lipid octafluoropropane gas core cationic microbubbles that could target the release of human Sirt3 reactivating the endogenous Sirt3 in hypertrophic hearts and protect against oxidative stress in a pig model of cardiac hypertrophy induced by aortic banding. Sirt3-CMBs may enhance cardiac diastolic function and ameliorate fibrosis and apoptosis. Our work provides a classical cationic lipid-based, UTMD-mediated Sirt3 delivery system for the treatment of Sirt3 in patients with established cardiac hypertrophy, as well as a promising therapeutic target to combat pathological cardiac hypertrophy.

Articular chondrocyte-derived extracellular vesicles promote cartilage differentiation of human umbilical cord mesenchymal stem cells by activation of autophagy.

BACKGROUND: Umbilical cord mesenchymal stem cell (HUCMSC)-based therapies were previously utilised for cartilage regeneration because of the chondrogenic potential of MSCs. However, chondrogenic differentiation of HUCMSCs is limited by the administration of growth factors like TGF-ß that may cause cartilage hypertrophy. It has been reported that extracellular vesicles (EVs) could modulate the phenotypic expression of stem cells. However, the role of human chondrogenic-derived EVs (C-EVs) in chondrogenic differentiation of HUCMSCs has not been reported. RESULTS: We successfully isolated C-EVs from human multi-finger cartilage and found that C-EVs efficiently promoted the proliferation and chondrogenic differentiation of HUCMSCs, evidenced by highly expressed aggrecan (ACAN), COL2A, and SOX-9. Moreover, the expression of the fibrotic marker COL1A and hypertrophic marker COL10 was significantly lower than that induced by TGF-ß. In vivo, C-EVs induced HUCMSCs accelerated the repair of the rabbit model of knee cartilage defect. Furthermore, C-EVs led to an increase in autophagosomes during the process of chondrogenic differentiation, indicating that C-EVs promote cartilage regeneration through the activation of autophagy. CONCLUSIONS: C-EVs play an essential role in fostering chondrogenic differentiation and proliferation of HUCMSCs, which may be beneficial for articular cartilage repair.

Identification of potential specific biomarkers and key signaling pathways between osteogenic and adipogenic differentiation of hBMSCs for osteoporosis therapy.

BACKGROUND: The differentiation of bone mesenchymal stem cells (BMSCs) into adipogenesis (AD) rather than osteogenesis (OS) is an important pathological feature of osteoporosis. Illuminating the detailed mechanisms of the differentiation of BMSCs into OS and AD would contribute to the interpretation of osteoporosis pathology. METHODS: To identify the regulated mechanism in lineage commitment of the BMSCs into OS and AD in the early stages, the gene expression profiles with temporal series were downloaded to reveal the distinct fates when BMSCs adopt a committed lineage. For both OS and AD lineages, the profiles of days 2-4 were compared with day 0 to screen the differentially expressed genes (DEGs), respectively. Next, the functional enrichment analysis was utilized to find out the biological function, and protein-protein interaction network to predict the central genes. Finally, experiments were performed to verify our finding. RESULTS: FoxO signaling pathway with central genes like FoxO3, IL6, and CAT is the crucial mechanism of OS, while Rap1 signaling pathway of VEGFA and FGF2 enrichment is more significant for AD. Besides, PI3K-Akt signaling pathway might serve as the latent mechanism about the initiation of differentiation of BMSCs into multiple lineages. CONCLUSION: Above hub genes and early-responder signaling pathways control osteogenic and adipogenic fates of BMSCs, which maybe mechanistic models clarifying the changes of bone metabolism in the clinical progress of osteoporosis. The findings provide a crucial reference for the prevention and therapy of osteoporosis.

Electrospun poly(3-hydroxybutyrate-co-4-hydroxybutyrate) /Octacalcium phosphate Nanofibrous membranes for effective guided bone regeneration.

Membranes used in guided bone regeneration (GBR) are required to exhibit high mechanical strength, biocompatibility, biodegradation, osteogenic and osteoinductive potential. In our study, poly(3-hydroxybutyrate-co-4-hydroxybutyrate)(P(3HB-co-4HB))/octacalcium phosphate (OCP) (P(3HB-co-4HB)/OCP) nanofibrous membranes were fabricated by electrospinning with two different P(3HB-co-4HB) to OCP ratios (P(3HB-co-4HB):OCP = 95:5 wt% and 90:10 wt%, termed P(3HB-co-4HB)/OCP(5)and P(3HB-co-4HB)/OCP (10), respectively) for GBR. The developed P(3HB-co-4HB)/OCP nanofibrous membranes were analysed for their osteogenic and osteoinductive properties using mesenchymal stem cells (MSCs) in vitro and in a calvarial bone defect rat model. The composite P(3HB-co-4HB)/OCP nanofibrous membranes showed decreased fibre size and enhanced tensile strength compared with those of P(3HB-co-4HB) nanofibrous membranes. In the in vitro studies, the P(3HB-co-4HB)/OCP membranes facilitated cell growth and osteoblastic differentiation of MSCs and were superior to P(3HB-co-4HB) membranes. After covered on the calvarial bone defects, P(3HB-co-4HB)/OCP membranes facilitated greater neobone formation than P(3HB-co-4HB) membranes did, as the result of histological evaluation and micro-CT analysis with higher bone volume/total volume (BV/TV) ratio and bone mineral density (BMD). P(3HB-co-4HB)/OCP(10) membranes with higher OCP content showed greater stiffness and osteoinductivity than P(3HB-co-4HB)/OCP (5)membranes, demonstrating the role of OCP in the composite membranes. These results indicated that electrospun P(3HB-co-4HB)/OCP nanofibrous membranes hold promise for the clinical application of GBR.

Salidroside enhances proliferation and maintains phenotype of articular chondrocytes for autologous chondrocyte implantation (ACI) via TGF-ß/Smad3 Signal.

Autologous chondrocyte implantation (ACI) is commonly used for the treatment of cartilage defects. Since the cell number for transplantation is limited, the expand culture of chondrocytes in vitro is needed. However, the phenotype of chondrocytes is easy to lose in monolayer cultured in vitro. Traditional growth factors such as transformation growth factor -ß1 (TGF-ß1) have been used for promoting the proliferation and maintained the phenotype of chondrocytes, but the high cost and functional heterogeneity limit their clinical application. It is of significant to develop substitutes that can accelerate proliferation and prevent dedifferentiation of chondrocytes for further study. In our present study, the effect of salidroside on proliferation and phenotype maintenance of chondrocytes and cartilage repair was investigated by performing the cell viability, morphology, glycosaminoglycan (GAG) synthesis, cartilage relative genes expression, macroscopic and histological analyzsis. The TGF-ß/smad3 signal which may involve in the protective effect of salidroside on chondrocytes was also detected by ELISA and qRT-PCR assays. The results indicated that salidroside could promote chondrocytes proliferation and enhance synthesis of cartilage extracellular matrix (ECM). Expression of collagen type I was significantly down-regulated which suggesting that salidroside could prevent chondrocytes from dedifferentiation. The in vivo experiments for cartilage repair also indicated that in the treatment of salidroside, chondrocytes used for ACI significantly accelerated the hyaline cartilage repair. While in the absence of salidroside, the repaired cartilage is mainly the fibrous cartilage. Additional experiments demonstrated that salidroside promotes the proliferation and maintain the phenotype of chondrocytes by activate the TGF-ß/smad3 signal. Salidroside may be a potential agent for ACI to promote the proliferation and maintain the phenotype of chondrocytes expansion in vitro.

Weak acidic stable carbazate modified cellulose membranes target for scavenging carbonylated proteins in hemodialysis.

Carbazate groups were grafted on the commercial cellulose membrane (CM) to specifically scavenge the carbonylated proteins for hemodialysis. It confirmed that carbazate groups were successfully covalently attached on the CMs by XPS and EDS, and the modified CMs still saved their original morphology and crystalline structures by SEM and XRD. Furthermore, the modified CMs presented favorable physicochemical stability at wide pH range from 2.5 to 7.4. It was also found that the carbazate modified CMs could selectively remove carbonylated proteins from acrolein treated bovine serum albumin (BSA) or ESRD patient's blood serum in PBS buffer. The modified CMs showed the potential to be utilized as the substitute of dialysis membranes in hemodialysis.

Syndecan-4 involves in the pathogenesis of rheumatoid arthritis by regulating the inflammatory response and apoptosis of fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and the pathogenesis of RA is still unknown. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) are of significance in the pathogenesis of RA. In this study, three microarray profiles (GSE55457, GSE55584, and GSE55235) of human joint FLSs from 33 RA patients and 20 normal controls were extracted from the Gene Expression Omnibus Dataset and analyzed to investigate the underlying pathogenesis of RA. As analyzed by the differently expressed genes, gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and protein-protein interaction network analysis, syndecan-4 (SDC4), a receptor of multiple cytokines and chemokines, which played a key role in the regulation of inflammatory response, was found to be an essential regulator in RA. To further validate these results, the levels of SDC4, reactive oxygen species (ROS), nitric oxide (NO), inflammation, and apoptosis in RA-FLSs were examined. SDC4-silenced RA-FLSs were also used. The results demonstrated that SDC4 and the level of ROS, NO, and inflammation were highly expressed while the apoptosis was decreased in RA-FLSs compared with normal FLSs. SDC4 silencing significantly suppressed the levels of ROS, NO, and inflammation; elevated the expression of nuclear factor erythroid 2-related factor 2; and promoted the apoptosis of RA-FLSs. Collectively, our results demonstrated a new mechanism of SDC4 in initiating the inflammation and inhibiting the apoptosis of RA-FLSs and that a potential target for the diagnosis and treatment of RA in the clinic might be developed.

BTN3A2 serves as a prognostic marker and favors immune infiltration in triple-negative breast cancer.

Immune infiltration is reported to be highly associated with tumor progress. Since butyrophilin subfamily 3 member A2 (BTN3A2) serves as a crucial mediator in immune activation, we aimed to investigate the correlation of BTN3A2 in immune infiltration and tumor prognosis via extensive-cancer analysis. The levels of BTN3A2 expression in extensive cancers were analyzed with Oncomine and TIMER databases. Univariate cox and multivariate cox regression analyses were conducted to assess the associations of BTN3A2 to prognosis of various cancers. The correlations of BTN3A2 with immune infiltration were assessed by TIMER database. It suggested that BTN3A2 was a potential prognosis signature for breast cancer (BRCA) and ovarian cancer (OV). However, immune infiltrations were highly correlated with BTN3A2 in triple-negative breast cancer (TNBC), compared with OV and other subtypes of BRCA. Multivariate cox regression analysis revealed that BTN3A2 was an independently prognostic signature of TNBC, as well as weighted correlation network analysis suggested BTN3A2 was only correlated with TNBC, rather than other subtypes of BRCA. Immune cell subtypes correlation analysis showed that BTN3A2 was highly correlated with general T, CD8+ T, T helper type 1, exhausted T cells, and dendritic cells in TNBC. And the coexpression geneset of BTN3A2 was mainly involved in T-cell receptor interaction and the nuclear factor-κB (NF-κB) signaling pathway. Collectively, BTN3A2 that was positively associated with better prognosis could be served as a special diagnostic and independently prognostic marker for TNBC by regulating the T-cell receptor interaction and NF-κB signaling pathways.

Implantable and degradable antioxidant poly(ε-caprolactone)-lignin nanofiber membrane for effective osteoarthritis treatment.

Osteoarthritis (OA) is one of the most common musculoskeletal disorders worldwide. Oxidative stress initiated by excessive free radicals such as reactive oxygen species (ROS) is a leading cause of cartilage degradation and OA. However, conventional injection or oral intake of antioxidants usually cannot provide effective treatment due to rapid clearance and degradation or low bioavailability. Here, a new strategy is proposed based on nanofibers made of poly (ε-caprolactone) (PCL) and PCL-grafted lignin (PCL-g-lignin) copolymer. Lignin offers intrinsic antioxidant activity while PCL tailors the mechanical properties. Electrospun PCL-lignin nanofibers show excellent antioxidant activity, low cytotoxicity and excellent anti-inflammatory effects as demonstrated using both H2O2-stimulated human chondrocytes and an OA rabbit model. PCL-lignin nanofibers inhibit ROS generation and activate antioxidant enzymes through autophagic mechanism. Arthroscopic implantation of nanofibrous membrane of PCL-lignin is effective to OA therapy because it is biocompatible, biodegradable and able to provide sustained antioxidant activity.

Electrospun PLGA/PCL/OCP nanofiber membranes promote osteogenic differentiation of mesenchymal stem cells (MSCs).

Nanofibers as niche-biomimetic scaffolds hold promise in guided bone regeneration (GBR). Here we fabricated poly (lactic-co-glycolic acid) (PLGA)/poly(caprolactone) (PCL)-doped octacalcium phosphate (OCP) nanofiber membranes via electrospinning and investigated the osteogenic behavior of marrow mesenchymal stem cells (MSCs) on the membranes. By adjusting different ratio of OCP to PLGA/PCL, three hybrid stents including PLGA/PCL, PLGA/PCL/2 wt%OCP, PLGA/PCL/4wt%OCP were successfully prepared. The PLGA/PCL/OCP membranes showed a decrease in fiber diameter compared with PLGA/PCL, leading to enhanced mechanical strength. In-vitro studies showed that PLGA/PCL/OCP membranes better supported cell adhesion, spreading and proliferation than PLGA/PCL. The incorporation of OCP via electrospinning also endowed the membranes with osteoinductive capacity, as evidenced by activation of ALP activity, increased gene expression of bone specific markers (such as Runx2, ALP, Col 1a1, OPN, OCN, BMP2), and mineral nodules formation compared to PLGA/PCL. Comparatively, PLGA/PCL/4wt%OCP showed better mechanical and biological performance than PLGA/PCL/2 wt%OCP, demonstrating the role of OCP in nanofiber membranes. Thus, the electrospun PLGA/PCL/OCP nanofiber membranes can be potentially developed as a promising hybrid stent for GBR.

Comparative profiling of chondrogenic differentiation of mesenchymal stem cells (MSCs) driven by two different growth factors.

This study aimed to investigate the mechanism of nerve growth factor (NGF) from cobra venom and human transforming growth factor-ß1 (TGF-ß1) on the chondrogenic induction of mesenchymal stem cells (MSCs). NGF and TGF-ß1 were used to induce chondrogenesis of MSCs from rabbits for 7 days. Total RNA was extracted for mRNA sequencing. Differentially expressed genes (DEGs), gene ontology (GO), KEGG pathway enrichment, and PPI network analysis were conducted to screen the specific signalling pathways and target genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to further confirm the relative target genes. The results showed that NGF could significantly promote the expression of hyaline cartilage specific genes (collagen type II alpha 1 chain, COL2A1) compared with TGF-ß1. PI3K-AKT signalling pathway is commonly involved in the chondrogenesis of MSCs induced by NGF and TGF-ß1. However, the expression levels of the genes in the PI3K-AKT signalling pathway were significantly higher in NGF group than that in the TGF-ß1 group. In the process of chondrogenesis of MSCs induced by NGF and TGF-ß1, integrin (ITGAs) were the targeted hub genes to activate the PI3K-AKT signalling pathway. NGF could activate more proliferation and differentiation genes in the process of chondrogenesis of MSCs than TGF-ß1. TGF-ß1 promoted angiogenesis by targeting the thrombospondin (THBS1) and THBS2 which might contribute to the osteophyte formation. PI3K-AKT was the crucial signalling pathway for chondrogenic differentiation. NGF could activate the PI3K-AKT signalling pathway to a higher level, and NGF had more specificity for promoting expression of specific genes of chondrocyte compared with TGF-ß1. SIGNIFICANCE OF THE STUDY: In our study, we compared two different growth factors in promoting cartilage differentiation of MSCs and found some similarities and differences. We revealed that both NGF and TGF-ß1 could activate the PI3K-AKT signalling pathway (the expression of it in NGF was higher) by targeting the ITGAs in the process of chondrogenesis from MSCs. However, NGF could activate more proliferation and differentiation genes in the process of chondrogenesis of MSCs, whereas TGF-ß1 caused osteophyte formation by activating THBS1 and THBS2. These might be the reason why NGF could promote cartilage differentiation more specifically.

Comparison of rheumatoid arthritis (RA) and osteoarthritis (OA) based on microarray profiles of human joint fibroblast-like synoviocytes.

The purpose of the present study was to investigate the underlying molecular mechanism of osteoarthritis (OA) and rheumatoid arthritis (RA) based on microarray profiles. Three human joint fibroblast-like synoviocytes (FLSs) microarray profiles including 26 OA samples, 33 RA samples, and 20 healthy control (HC) samples were downloaded from the GEO database. Differentially expressed genes (DEGs) between OA and HC (DEGsOA) and RA and HC (DEGsRA) were identified. Co-expressed and specific genes were analysed between DEGsOA and DEGsRA. Gene ontology, KEGG pathway enrichment, PPI network, and GSEA were performed to predict the function of DEGs. Two hundred seventy-six and 410 differential genes in DEGsOA and DEGsRA were observed. One hundred fifty coexpressed genes and 126 OA-specific genes (SELE, SERPINE1, and NFKBIA were the key genes) between DEGsOA and DEGsRA were enriched in the tumour necrosis factor (TNF) signalling pathway. However, 260 RA-specific genes of which the key genes were CCR5, CCR7, CXCR4, CCL5, and CCR4 were enriched in chemokine signalling pathway. Therefore, FLSs might exert an inflammatory effect by regulating TNF signalling pathway, targeting SELE, SERPINE1, and NFKBIA during the process of OA. Although TNF signalling pathway was also involved in the synovitis of RA, chemokine signalling pathway played the key role in RA FLSs mediating cell migration, invasion, and release of chemotaxis. In addition, CCR5, CCR7, CXCR4, CCL5, and CCR4 might be hub genes in RA. The different biomarkers and pathways identified in OA and RA may provide references for further study. SIGNIFICANCE OF THE STUDY: This study revealed the similar and different mechanisms of FLSs and different biomarkers that might with important regulatory effects on RA and OA. In OA, FLSs played an inflammatory role through TNF signalling pathway, targeting SELE, SERPINE1, and NFKBIA. Although TNF signalling pathway was also involved in the synovitis of RA, chemokine signalling pathway was a crucial pathway in mediating FLSs migration, invasion, and release of chemotaxis. CCR5, CCR7, CXCR4, CCL5, and CCR4 might be keys genes in RA. We expect that our results will bring more comprehensively understanding between RA and OA for researchers.