Anabolic Genes Research Articles

Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing

PurposeThe study aimed to compare the effect of mincing bovine articular cartilage with different shaver blades on chondrocyte viability.MethodsBovine articular cartilage was harvested either with a scalpel or with three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage harvested with a scalpel was then minced into fragments smaller than 1 mm3 with a scalpel. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, the following measurements were performed: metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1 and ACAN) and catabolic genes (MMP1 and MMP13), live/dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes.ResultsMincing the cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p < 0.001). Gene expression of anabolic genes (COL2A1 and ACAN) was reduced, while catabolic genes (MMP1 and MMP13) were increased after day 7 in all shaver conditions. Confocal microscopy showed a thin line of dead cells at the lesion side with viable cells beneath for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p < 0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing.ConclusionMincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing immediately after harvest and after seven days in culture. This suggests that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy.Level of evidenceLevel II therapeutic study.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24°C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

γ-Aminobutyric acid treatment induced chilling tolerance in postharvest kiwifruit (Actinidia chinensis cv. Hongyang) via regulating ascorbic acid metabolism

The effect of γ-Aminobutyric acid (GABA) treatment on ascorbic acid (AsA) metabolism and chilling injury in postharvest kiwifruit was studied. The results revealed that kiwifruit treated with GABA displayed higher chilling tolerance and better quality maintenance as compared to the controls. Higher AsA was observed in GABA-treated fruit which was beneficial to cell membrane protection and damage alleviation against chilling mediated oxidative stress. Gene expression analysis found the increased expression of AsA anabolic and regenerative genes and down-regulation of its catabolic genes together could contribute to the elevation of AsA levels in kiwifruit after GABA treatment. In addition, the transcripts of several candidate transcription factors such as bHLHs and HZ1 involved in AsA biosynthesis were also enhanced by GABA treatment. Collectively, our results indicated that GABA induced chilling tolerance in postharvest kiwifruit due to the higher AsA content by positively regulating ascorbate metabolic genes and candidate transcription factors.

GATA-type transcriptional factor SpGAT1 interacts with SpMIG1 and promotes lipid accumulation in the oleaginous yeast Saitozyma podzolica zwy-2-3

BackgroundIn oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in Saitozyma podzolica zwy-2-3.ResultsCompared with WT, Delta gat1, and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of Delta gat1 decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation.ConclusionOur findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

Improving nitrogen and phosphorus removal and sludge reduction in new integrated sewage treatment facility by adjusting biomass concentration

Aiming at the practical problem of rural wastewater and excess sludge treatment difficulties, micro-pressure swirl reactor (MPSR) was employed to carry out the experiment on the treatment of sewage under five different biomass concentration (MLSS concentration) conditions (4.14, 6.05, 7.98, 11.45, 14.46 g/L). The results showed that the nitrogen removal and phosphorus removal and sludge reduction performance of the system showed an increase and then a decrease with increasing MLSS concentration, and 7.98–11.45 g/L was the optimal MLSS concentration range. In addition, parameters related to in situ sludge reduction were further examined, and the extracellular polymeric substance content and sludge dehydrogenase activity were negatively and positively correlated with sludge reduction, respectively. According to the microbial analysis, at suitable MLSS concentration, beneficial microorganisms (Tetrasphaera, Aeromonas, Candidatus_Competibacter) were enriched in the MPSR to facilitate the nitrogen and phosphorus removal and sludge reduction of the system. In addition, the potential gene(nirK, norB, nosZ) functions of the microorganisms were positively regulated. The increase in the expression abundance ratio of dissimilatory and anabolic nitrate reduction genes played a positive role in denitrification and sludge reduction of the system.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

In vitro study. To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. hNPCs were exposed to 5, 10, and 25ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1β gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10ng/mL IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with irisin). Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1β mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1β levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1β and ADAMTS-5 levels. Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

Macrophage polarization regulates intervertebral disc degeneration by modulating cell proliferation, inflammation mediator secretion, and extracellular matrix metabolism.

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1β, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.

Water temperature affects the protein requirements, growth performance, and nutritional metabolism of grass carp (Ctenopharyngodon idella) juveniles

This study aimed to determine whether water temperature and dietary protein levels played a role in regulating the growth and nutritional metabolism of juvenile grass carp (4.47 ± 0.01 g). Six dietary protein levels (18.20%, 24.32%, 30.60%, 36.42%, 43.27%, 49.49%) and two levels of water temperature (23 ℃ and 28 ℃) were used with a 6 × 2 experimental design method. After 60 days, the results demonstrated that appropriate dietary protein levels (30.60%, 23 ℃; 36.42%, 28 ℃) significantly increased the specific growth rate and weight gain rate of grass carp and significantly decreased the feed conversion ratio (P<0.05), and grass carp at 28 °C showed better feed utilization efficiency and growth performance. In addition, grass carp at 28 °C showed higher metabolic levels, and the expression of genes related to protein synthesis/catabolism, lipid synthesis/catabolism, and gluconeogenesis was significantly higher than at 23 °C. The highest plasma total cholesterol, plasma glucose and whole-body lipid content was observed in grass carp reared at 28 °C under low dietary protein level feeding (18.20%), while similar phenomena appeared in the high dietary protein levels (43.27%, 49.49%) feeding group at 23 °C. Correspondingly, a significantly higher the expression level of lipid anabolism genes was also observed in this dietary protein level. In addition, the plasma total cholesterol, total triglyceride, glucose, and total protein contents of grass carp under 23 °C culture were significantly higher than those of the 28 °C group. Based on the feed conversion ratio and specific growth rate by regression lines calculated using a quadratic model, the protein requirements of juvenile grass carp reared at 28 °C were 38.31%, and 38.03%, which were higher than the protein requirements of 36.76%, and 34.13% at 23 °C. These results may provide theoretical guidance for the precise protein requirements for grass carp juveniles reared at different temperatures.

Increased Chondroprotective Effect of Combining Hyaluronic Acid with a Glucocorticoid Compared to Separate Administration on Cytokine-Treated Osteoarthritic Chondrocytes in a 2D Culture

Intra-articular injections of glucocorticoids (GC) or hyaluronic acid (HA) are commonly used interventions for patients suffering from knee osteoarthritis (OA). Both substances are combined to achieve a chondroprotective and anti-inflammatory effect. Clinical studies have shown benefits, but data on the cellular level are still lacking. This study aimed to investigate the effect of the GC triamcinolone hexacetonide, HA, and a mix of both substances on cytokine-treated chondrocytes in vitro. Chondrocytes isolated from human articular cartilage were seeded on 6- and 24-well plates. Mimicking OA’s inflammatory state, cells were treated with IL-1β and IL-17 for six days, whereby, after three days, test substances (10%) were added to the culture medium. Chondrocytes were analyzed on days three and six concerning their actin polymerization, expression of anabolic and catabolic genes, metabolic activity, cytokine release, and reactive oxygen species (ROS). Adding HA or GC/HA to the inflammatory culture medium increased the metabolic activity of chondrocytes, while groups containing GC reduced catabolic gene expression and the release of TNF-α. In addition, enhanced F-actin content was shown supplementing HA or GC/HA to the culture medium. Supplementing GC with HA leads to an anti-inflammatory and chondroprotective effect by diminishing the side effects of GC supplementation alone.

First study on the effect of transforming growth factor beta 1 and insulin-like growth factor 1 on the chondrogenesis of elephant articular chondrocytes in a scaffold-based 3D culture model.

Background and Aim:Osteoarthritis (OA) is recognized as a degenerative joint disease that leads to chronic pain and low quality of life in animals. Captive elephants, the largest land mammals with a long lifespan, are more prone to develop OA due to restricted spaces and insufficient physical activity. This study aimed to investigate the effect of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) on elephant chondrogenesis in a scaffold culture of articular chondrocytes.Materials and Methods:Elephant chondrocytes-seeded gelatin scaffolds were cultured in chondrogenic media with or without 10 ng/mL of TGF-β1 or IGF-1 alone or 5–10 ng/mL of their combination for up to 21 days. The mRNA expression of cartilage-specific anabolic genes, ACAN and COL2A1, was analyzed using a real-time reverse transcription-polymerase chain reaction. The amounts of sulfated glycosaminoglycans (sGAGs) in conditioned media and contents in cultured scaffolds were determined through dimethylmethylene blue assay. Cell morphology, accumulation of proteoglycans, and details of the cultured scaffolds were determined using hematoxylin-eosin staining, safranin O staining, and scanning electron microscopy (SEM), respectively.Results:TGF-β1 alone significantly upregulated ACAN gene expression but not COL2A1, while IGF-1 alone did not enhance both ACAN and COL2A1 genes. The combination significantly upregulated both mRNA expression levels of ACAN and COL2A1 gene at day 14. The sGAGs accumulation and contents in the treatment groups, except IGF-1 tended to be higher than the controls, concomitantly with the production of the extracellular matrix, showed the formation of a cartilage-like tissue through histological and SEM analyses.Conclusion:Together, our results suggest that the single treatment of TGF-β1 has a selective effect on ACAN gene, while the combined growth factors seem to be an advantage on elephant chondrogenesis. This three-dimensional culture model is probably helpful for developing cartilage regeneration in vitro and is further applied in tissue engineering for OA treatment in vivo.

Self-Assembled Nanospheres with Enhanced Interfacial Lubrication for the Treatment of Osteoarthritis

Osteoarthritis is associated with an increase in mechanical friction of the joint, which causes irreversible damage to articular cartilage. Consequently, it is crucial to restore joint lubrication for effectively treating osteoarthritis. In the present study, hyaluronic acid (HA)-based zwitterionic nanospheres with phosphocholine groups on the surface were synthesized, which achieved excellent lubrication behavior due to the hydration lubrication mechanism. Specifically, HA was initially thiolated and modified with hexadecylamine based on an amidation reaction, then it was grafted with 2-methacryloyloxyethyl phosphocholine (MPC) by the thiol-ene click reaction, and finally self-assembled into nanospheres (HA-MPC) by hydrophobic interaction and cross-linking of the thiol group. The lubrication test demonstrated that the HA-MPC nanospheres improved lubrication under shear force, with a 40% reduction in the friction coefficient compared with HA. The in vitro experiment indicated that the HA-MPC nanospheres had excellent biocompatibility, and they upregulated the cartilage anabolic gene and downregulated cartilage catabolic proteases as well as the pain-related gene. The in vivo test showed that the injection of HA-MPC nanospheres to the joint cavity could inhibit the development of osteoarthritis, which was examined based on histological staining and also morphological evaluation. In conclusion, the new self-assembled zwitterionic HA-MPC nanospheres may be intra-articularly injected for the effective treatment of osteoarthritis by restoring joint lubrication.

IL-1Ra deficiency accelerates intervertebral disc degeneration in C57BL6J mice.

The expression of Interleukin‐1ß (IL‐1ß) and its antagonist and Interleukin‐1 receptor antagonist (IL‐1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL‐1β activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL‐1β activity on intervertebral disc in mice with an IL‐1Ra gene deletion.IL‐1Ra−/− mice and wild‐type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL‐1β pathways. To test differences in injury response, pinprick annular puncture was performed on IL‐1Ra−/− and WT mice and evaluated similarly.IL‐1Ra−/− IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL‐1Ra−/− IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL‐1Ra−/− IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2‐week and 6‐week post‐injured WT and IL1‐Ra−/− IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL‐1Ra−/− mice at 2 weeks, returning by 6 weeks post injury.In the present study, IL‐1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro‐inflammatory cytokine gene expression. The histopathology of IL‐1Ra−/− IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra−/− on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL‐1β signaling in IVD pathobiology. Despite a pro‐inflammatory environment, the annular puncture was no worse in IL‐1Ra−/− mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL‐1β‐driven inflammation is important in IVD degeneration.

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence

BackgroundOsteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear. PurposeTo experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism. MethodsUPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZDʹs efficacy and safety in OA patients. ResultsThe UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients. ConclusionThe scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading.

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

Inhibition of the extracellular signal-regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells.

Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-αand the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036)were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.

Current Knowledge and Future Therapeutic Prospects in Symptomatic Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is the main source of intractable lower back pain, and symptomatic IVD degeneration could be due to different degeneration mechanisms. In this article, we describe the molecular basis of symptomatic IVD degenerative disc diseases (DDDs), emphasizing the role of degeneration, inflammation, angiogenesis, and extracellular matrix (ECM) regulation during this process. In symptomatic DDD, pro-inflammatory mediators modulate catabolic reactions, resulting in changes in ECM homeostasis and, finally, neural/vascular ingrowth-related chronic intractable discogenic pain. In ECM homeostasis, anabolic protein-regulating genes show reduced expression and changes in ECM production, while matrix metalloproteinase gene expression increases and results in aggressive ECM degradation. The resultant loss of normal IVD viscoelasticity and a concomitant change in ECM composition are key mechanisms in DDDs. During inflammation, a macrophage-related cascade is represented by the secretion of high levels of pro-inflammatory cytokines, which induce inflammation. Aberrant angiogenesis is considered a key initiative pathologic step in symptomatic DDD. In reflection of angiogenesis, vascular endothelial growth factor expression is regulated by hypoxia-inducible factor-1 in the hypoxic conditions of IVDs. Furthermore, IVD cells undergoing degeneration potentially enhance neovascularization by secreting large amounts of angiogenic cytokines, which penetrate the IVD from the outer annulus fibrosus, extending deep into the outer part of the nucleus pulposus. Based on current knowledge, a multi-disciplinary approach is needed in all aspects of spinal research, starting from basic research to clinical applications, as this will provide information regarding treatments for DDDs and discogenic pain.

Transcriptional Integration of Distinct Microbial and Nutritional Signals by the Small Intestinal Epithelium.

Background & AimsThe intestine constantly interprets and adapts to complex combinations of dietary and microbial stimuli. However, the transcriptional strategies by which the intestinal epithelium integrates these coincident sources of information remain unresolved. We recently found that microbiota colonization suppresses epithelial activity of hepatocyte nuclear factor 4 nuclear receptor transcription factors, but their integrative regulation was unknown.MethodsWe compared adult mice reared germ-free or conventionalized with a microbiota either fed normally or after a single high-fat meal. Preparations of unsorted jejunal intestinal epithelial cells were queried using lipidomics and genome-wide assays for RNA sequencing and ChIP sequencing for the activating histone mark H3K27ac and hepatocyte nuclear factor 4 alpha.ResultsAnalysis of lipid classes, genes, and regulatory regions identified distinct nutritional and microbial responses but also simultaneous influence of both stimuli. H3K27ac sites preferentially increased by high-fat meal in the presence of microbes neighbor lipid anabolism and proliferation genes, were previously identified intestinal stem cell regulatory regions, and were not hepatocyte nuclear factor 4 alpha targets. In contrast, H3K27ac sites preferentially increased by high-fat meal in the absence of microbes neighbor targets of the energy homeostasis regulator peroxisome proliferator activated receptor alpha, neighbored fatty acid oxidation genes, were previously identified enterocyte regulatory regions, and were hepatocyte factor 4 alpha bound.ConclusionsHepatocyte factor 4 alpha supports a differentiated enterocyte and fatty acid oxidation program in germ-free mice, and that suppression of hepatocyte factor 4 alpha by the combination of microbes and high-fat meal may result in preferential activation of intestinal epithelial cell proliferation programs. This identifies potential transcriptional mechanisms for intestinal adaptation to multiple signals and how microbiota may modulate intestinal lipid absorption, epithelial cell renewal, and systemic energy balance.

Dynamic hyaluronic acid hydrogel with covalent linked gelatin as an anti-oxidative bioink for cartilage tissue engineering

In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytes in vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibility in vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.

A human in vitro 3D neo-cartilage model to explore the response of OA risk genes to hyper-physiological mechanical stress

ObjectiveDue to the complexity and heterogeneity of osteoarthritis (OA) pathophysiology, studying the interaction between intrinsic molecular changes in chondrocytes after hyper-physiological mechanical stress (MS) and aberrant signalling of OA risk genes remains a challenge. In this study we set out to set up an in vitro 3D neo cartilage pellet model that enables us to explore the responses of OA risk genes to hyper-physiological MS. DesignHuman primary chondrocyte neo-cartilage pellets were exposed for 2 days to 2 ​× ​10 ​min of hyper-physiological dynamic MS attained by a 20% strain and a frequency of 5 ​Hz. In order to assess cartilage damage, sulphated glycosaminoglycan (sGAG) content in the neo-cartilage was quantified using Alcian blue staining and a dimethyl methylene blue (DMMB) assay, while cleavage of aggrecan was visualized by immunohistochemical staining of aggrecan neo-epitope NITEGE. In addition, changes in expression levels of catabolic, anabolic and hypertrophic genes, and of three OA risk genes; IL11, MGP and TGFA were determined. ResultsHyper-physiological MS induced cartilage damage, as reflected by decreased sGAG content. mRNA levels of aggrecanase ADAMTS5 were increased, while hypertrophic gene RUNX2 was downregulated. MS increased expression of pro-apoptotic marker NOXA. Furthermore, 20% MS led to increased expression of all three OA risk genes IL11, MGP and TGFA. ConclusionsWe established a human in vitro model in which hyper-physiological MS induced cartilage damage and catabolic signalling. Next, we demonstrated its usage to study OA risk genes and their response to the mechanical aspects of OA pathophysiology.

Establishment of an Ex Vivo Inflammatory Osteoarthritis Model With Human Osteochondral Explants.

Osteoarthritis (OA) is the most common degenerative joint disease without clear pathophysiological mechanism and effective drugs for treatment. Although various animal models exist, the translation of the outcome into clinics remains difficult due to species differences. In this study, an ex vivo inflammatory OA model was induced using different concentrations of interleukin one beta (IL-1β) and tumor necrosis factor α (TNF-α) on explants from the human femoral head. In the inflammatory OA groups, the gene expression levels of cartilage catabolism (matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3)), and inflammation (interleukin 6 (IL-6), interleukin 8 (IL-8)) markers were significantly upregulated, while the anabolic genes (collagen 2 (COL2), aggrecan (ACAN), and proteoglycan 4 (PRG4)) were downregulated compared to the control group. The release of cytokines (IL-6, IL-8) and nitric oxide (NO) in the conditioned medium was also upregulated in inflammatory OA groups. The Safranin O/Fast Green staining showed loss of proteoglycan in the superficial zone cartilage after cytokine treatment. The results indicated that an ex vivo inflammation and degeneration model was successfully established using osteochondral explants from the human femoral head. This model can be used to elucidate the in-depth mechanism of inflammatory OA and to screen new drugs for OA treatment.