Microfluidic human physiomimetic liver model as a screening platform for drug induced liver injury.

The pre-clinical animal models often fail to predict intrinsic and idiosyncratic drug induced liver injury (DILI), thus contributing to drug failures in clinical trials, black box warnings and withdrawal of marketed drugs. This suggests a critical need for human-relevant in vitro models to predict diverse DILI phenotypes. In this study, a porcine liver extracellular matrix (ECM) based biomaterial ink with high printing fidelity, biocompatibility and tunable rheological and mechanical properties is formulated for supporting both parenchymal and non-parenchymal cells. Further, we applied 3D printing and microfluidic technology to bioengineer a human physiomimetic liver acinus model (HPLAM), recapitulating the radial hepatic cord-like structure with functional sinusoidal microvasculature network, biochemical and biophysical properties of native liver acinus. Intriguingly, the human derived hepatic cells incorporated HPLAM cultured under physiologically relevant microenvironment, acts as metabolic biofactories manifesting enhanced hepatic functionality, secretome levels and biomarkers expression over several weeks. We also report that the matured HPLAM reproduces dose- and time-dependent hepatotoxic response of human clinical relevance to drugs typically recognized for inducing diverse DILI phenotypes as compared to conventional static culture. Overall, the developed HPLAM emulates in vivo like functions and may provide a useful platform for DILI risk assessment to better determine safety and human risk.

Vitamin B6 alleviates osteoarthritis by suppressing inflammation and apoptosis.

BACKGROUND: Although various anti-inflammatory medicines are widely recommended for osteoarthritis (OA) treatment, no significantly clinical effect has been observed. This study aims to examine the effects of vitamin B6, a component that has been reported to be capable of alleviating inflammation and cell death in various diseases, on cartilage degeneration in OA. METHODS: Collagen-induced arthritis (CIA) mice model were established and the severity of OA in cartilage was determined using the Osteoarthritis Research Society International (OARSI) scoring system. The mRNA and protein levels of indicators associated with extracellular matrix (ECM) metabolism, apoptosis and inflammation were detected. The effect of vitamin B6 (VB6) on the mice were assessed using HE staining and masson staining. The apoptosis rate of cells was assessed using TdT-mediated dUTP nick end labeling. RESULTS: Our results showed a trend of improved OARSI score in mice treated with VB6, which remarkably inhibited the hyaline cartilage thickness, chondrocyte disordering, and knees hypertrophy. Moreover, the VB6 supplementation reduced the protein expression of pro-apoptosis indicators, including Bax and cleaved caspase-3 and raised the expression level of anti-apoptosis marker Bcl-2. Importantly, VB6 improved ECM metabolism in both in vivo and in vitro experiments. CONCLUSIONS: This study demonstrated that VB6 alleviates OA through regulating ECM metabolism, inflammation and apoptosis in chondrocytes and CIA mice. The findings in this study provide a theoretical basis for targeted therapy of OA, and further lay the theoretical foundation for studies of mechanisms of VB6 in treating OA.

Schiff base crosslinked hyaluronic acid hydrogels with tunable and cell instructive time-dependent mechanical properties.

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.

ECM-mimetic, NSAIDs loaded thermo-responsive, immunomodulatory hydrogel for rheumatoid arthritis treatment.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, and it leads to irreversible inflammation in intra-articular joints. Current treatment approaches for RA include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), corticosteroids, and biological agents. To overcome the drug-associated toxicity of conventional therapy and transdermal tissue barrier, an injectable NSAID-loaded hydrogel system was developed and explored its efficacy. RESULTS: The surface morphology and porosity of the hydrogels indicate that they mimic the natural ECM, which is greatly beneficial for tissue healing. Further, NSAIDs, i.e., diclofenac sodium, were loaded into the hydrogel, and the in vitro drug release pattern was found to be burst release for 24 h and subsequently sustainable release of 50% drug up to 10 days. The DPPH assay revealed that the hydrogels have good radical scavenging activity. The biocompatibility study carried out by MTT assay proved good biocompatibility and anti-inflammatory activity of the hydrogels was carried out by gene expression study in RAW 264.7 cells, which indicate the downregulation of several key inflammatory genes such as COX-2, TNF-α & 18s. CONCLUSION: In summary, the proposed ECM-mimetic, thermo-sensitive in situ hydrogels may be utilized for intra-articular inflammation modulation and can be beneficial by reducing the frequency of medication and providing optimum lubrication at intra-articular joints.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Phillyrin reduces ROS production to alleviate the progression of intervertebral disc degeneration by inhibiting NF-κB pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is an increasingly important cause of low back pain (LBP) that results in substantial health and economic burdens. Inflammatory pathway activation and the production of reactive oxygen species (ROS) play vital roles in the progression of IDD. Several studies have suggested that phillyrin has a protective role and inhibits inflammation and the production of ROS. However, the role of phillyrin in IDD has not been confirmed. PURPOSE: The purpose of this study was to investigate the role of phillyrin in IDD and its mechanisms. STUDY DESIGN: To establish IDD models in vivo, ex-vivo, and in vitro to verify the function of phillyrin in IDD. METHOD: The effects of phillyrin on extracellular matrix (ECM) degeneration, inflammation, and oxidation in nucleus pulposus (NP) cells were assessed using immunoblotting and immunofluorescence analysis. Additionally, the impact of phillyrin administration on acupuncture-mediated intervertebral disc degeneration (IDD) in rats was evaluated using various techniques such as MRI, HE staining, S-O staining, and immunohistochemistry (IHC). RESULT: Pretreatment with phillyrin significantly inhibited the IL-1ß-mediated reduction in the degeneration of ECM and apoptosis by alleviating activation of the NF-κB inflammatory pathway and the generation of ROS. In addition, in vivo and ex-vivo experiments verified the protective effect of phillyrin against IDD. CONCLUSION: Phillyrin can attenuate the progression of IDD by reducing ROS production and activating inflammatory pathways.

Inhibitory effect of doxycycline conjugated with deoxycholic acid and polyethylenimine conjugate on nasal fibroblast differentiation and extracellular production.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting the sinuses or nose. Persistent inflammatory responses can lead to tissue remodeling, which is a pathological characteristics of CRS. Activation of fibroblasts in the nasal mucosal stroma, differentiation and collagen deposition, and subepithelial fibrosis have been associated with CRS. OBJECTIVES: We aimed to assess the inhibitory effects of doxycycline and deoxycholic acid-polyethyleneimine conjugate (DA3-Doxy) on myofibroblast differentiation and extracellular matrix (ECM) production in nasal fibroblasts stimulated with TGF-ß1. METHODS: To enhance efficacy, we prepared DA3-Doxy using a conjugate of low-molecular-weight polyethyleneimine (PEI) (MW 1800) and deoxycholic acid (DA) and Doxy. The synthesis of the DA3-Doxy polymer was confirmed using nuclear magnetic resonance, and the critical micelle concentration required for cationic micelle formation through self-assembly was determined. Subsequently, the Doxy loading efficiency of DA3 was assessed. The cytotoxicity of Doxy, DA3, PEI, and DA-Doxy in nasal fibroblasts was evaluated using the WST-1 assay. The anti-tissue remodeling and anti-inflammatory effects of DA3-Doxy and DA3 were examined using real-time polymerase chain reaction (Real-time PCR), immunocytochemistry, western blot, and Sircol assay. RESULTS: Both DA3 and DA3-Doxy exhibited cytotoxicity at 10 µg/ml in nasal fibroblasts. Doxy partially inhibited α-smooth muscle actin, collagen types I and III, and fibronectin. However, DA3-Doxy significantly inhibited α-SMA, collagen types I and III, and fibronectin at 5 µg/ml. DA3-Doxy also modulated TGF-ß1-induced changes in the expression of MMP 1, 2, and 9. Nonetheless, TGF-ß1-induced expression of MMP3 was further increased by DA3-Doxy. The expression of TIMP 1 and 2 was partially reduced with 5 µg/ml DA3-Doxy. CONCLUSIONS: Although initially developed for the delivery of genetic materials or drugs, DA3 exhibits inhibitory effects on myofibroblast differentiation and ECM production. Therefore, it holds therapeutic potential for CRS, and a synergistic effect can be expected when loaded with CRS treatment drugs.

1-Methyltryptophan treatment ameliorates high-fat diet-induced depression in mice through reversing changes in perineuronal nets.

Depression and obesity are prevalent disorders with significant public health implications. In this study, we used a high-fat diet (HFD)-induced obese mouse model to investigate the mechanism underlying HFD-induced depression-like behaviors. HFD-induced obese mice exhibited depression-like behaviors and a reduction in hippocampus volume, which were reversed by treatment with an indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-MT). Interestingly, no changes in IDO levels were observed post-1-MT treatment, suggesting that other mechanisms may be involved in the anti-depressive effect of 1-MT. We further conducted RNA sequencing analysis to clarify the potential underlying mechanism of the anti-depressive effect of 1-MT in HFD-induced depressive mice and found a significant enrichment of shared differential genes in the extracellular matrix (ECM) organization pathway between the 1-MT-treated and untreated HFD-induced depressive mice. Therefore, we hypothesized that changes in ECM play a crucial role in the anti-depressive effect of 1-MT. To this end, we investigated perineuronal nets (PNNs), which are ECM assemblies that preferentially ensheath parvalbumin (PV)-positive interneurons and are involved in many abnormalities. We found that HFD is associated with excessive accumulation of PV-positive neurons and upregulation of PNNs, affecting synaptic transmission in PV-positive neurons and leading to glutamate-gamma-aminobutyric acid imbalances in the hippocampus. The 1-MT effectively reversed these changes, highlighting a PNN-related mechanism by which 1-MT exerts its anti-depressive effect.

Paeoniflorin alleviates TGF-ß2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

BACKGROUND: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-ß2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. METHODS: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-ß2 (5 ng/mL). PAE (300 µM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-ß2 for 22 h. SB-431542, a TGF-ß receptor inhibitor (10 µM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-ß2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. RESULTS: PAE attenuated TGF-ß2-induced oxidative stress and suppressed TGF-ß2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-ß2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-ß2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. CONCLUSION: PAE alleviates TGF-ß2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Extracellular Matrix-Mimetic Intrinsic Versatile Coating Derived from Marine Adhesive Protein Promotes Diabetic Wound Healing through Regulating the Microenvironment.

The management of diabetic wound healing remains a severe clinical challenge due to the complicated wound microenvironments, including abnormal immune regulation, excessive reactive oxygen species (ROS), and repeated bacterial infections. Herein, we report an extracellular matrix (ECM)-mimetic coating derived from scallop byssal protein (Sbp9Δ), which can be assembled in situ within 30 min under the trigger of Ca2+ driven by strong coordination interaction. The biocompatible Sbp9Δ coating and genetically programmable LL37-fused coating exhibit outstanding antioxidant, antibacterial, and immune regulatory properties in vitro. Proof-of-concept applications demonstrate that the coating can reliably promote wound healing in animal models, including diabetic mice and rabbits, ex vivo human skins, and Staphylococcus aureus-infected diabetic mice. In-depth mechanism investigation indicates that improved wound microenvironments accelerated wound repair, including alleviated bacterial infection, lessened inflammation, appearance of abundant M2-type macrophages, removal of ROS, promoted angiogenesis, and re-epithelialization. Collectively, our investigation provides an in situ, convenient, and effective approach for diabetic wound repair.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro.

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

Vitamin K2 ameliorates osteoarthritis by suppressing ferroptosis and extracellular matrix degradation through activation GPX4's dual functions.

Vitamin K2 (VK2) is an effective compound for anti-ferroptosis and anti-osteoporosis, and Semen sojae praeparatum (Dandouchi in Chinese) is the main source of VK2. Chondrocyte ferroptosis and extracellular matrix (ECM) degradation playing a role in the pathogenesis of osteoarthritis (OA). Glutathione peroxidase 4 (GPX4) is the intersection of two mechanisms in regulating OA progression. But no studies have elucidated the therapeutic effects and mechanisms of VK2 on OA. This study utilized an in vivo rat OA model created via anterior cruciate ligament transection (ACLT) and an in vitro chondrocyte oxidative damage model induced by TBHP to investigate the protective effects and mechanisms of action of VK2 in OA. Knee joint pain in mice was evaluated using the Von Frey test. Micro-CT and Safranin O-Fast Green staining were employed to observe the extent of damage to the tibial cartilage and subchondral bone, while immunohistochemistry and PCR were used to examine GPX4 levels in joint cartilage. The effects of VK2 on rat chondrocyte viability were assessed using CCK-8 and flow cytometry assays, and chondrocyte morphology was observed with toluidine blue and alcian blue staining. The impact of VK2 on intracellular ferroptosis-related markers was observed using fluorescent staining and flow cytometry. Protein expression changes were detected by immunofluorescence and Western blot analysis. Furthermore, specific protein inhibitors were applied to confirm the dual-regulatory effects of VK2 on GPX4. VK2 can increase bone mass and cartilage thickness in the subchondral bone of the tibia, and reduce pain and the OARSI score induced by OA. Immunohistochemistry results indicate that VK2 exerts its anti-OA effects by regulating GPX4 to delay ECM degradation. VK2 can inhibit the activation of the MAPK/NFκB signaling pathway caused by reduced expression of intracellular GPX4, thereby decreasing ECM degradation. Additionally, VK2 can reverse the inhibitory effect of RSL3 on GPX4, increase intracellular GSH content and the GSH/GSSG ratio, reduce MDA content, and rescue chondrocyte ferroptosis. The protective mechanism of VK2 may involve its dual-target regulation of GPX4, reducing chondrocyte ferroptosis and inhibiting the MAPK/NFκB signaling pathway to decelerate the degradation of the chondrocyte extracellular matrix.

17ß-estradiol activates SOX6 to balance the anabolism and catabolism via estrogen receptor 2 in chondrocyte.

We investigated the influence of 17ß-estradiol (17ß-E2) on cartilage extracellular matrix (ECM) homeostasis in postmenopausal women. We focused on the roles of estrogen receptors (ESR) and SOX6 in 17ß-E2-mediated stimulation of ECM metabolism during chondrocyte (CH) degeneration. We compared the expression of anabolic genes (collagen II and aggrecan) and catabolic genes (MMPs and TIMPs) in IL-1ß-induced CH degeneration in vitro, with and without 17ß-E2 supplementation. We separately silenced the SOX6, ESR1, and ESR2 genes in CHs to determine their impact on 17ß-E2 treatment. Additionally, we used Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and luciferase assays to investigate protein-DNA interactions within ESR2 and SOX6-promoter complexes. After three days of IL-1ß treatment, ESR1/2, SOX6, collagen II, aggrecan, and TIMP1/3 were decreased, while MMP3/9/13 were increased. The addition of 17ß-E2 partially reversed these effects, but silencing SOX6, ESR1, or ESR2 weakened the protective effects of 17ß-E2. Silencing ESR2, but not ESR1, abolished the upregulation of SOX6 induced by 17ß-E2. ESR2 was found to bind the SOX6 promoter and regulate SOX6 expression. 17ß-E2 upregulates SOX6 through ESR2 mediation, and the synergistic effect of 17ß-E2 and ESR2 on SOX6 balances ECM metabolism in CHs.

Intratumor transforming growth factor-ß signaling with extracellular matrix-related gene regulation marks chemotherapy-resistant gastric cancer.

Drug-tolerant persister (DTP) cells remain following chemotherapy and can cause cancer relapse. However, it is unclear when acquired resistance to chemotherapy emerges. Here, we compared the gene expression profiles of gastric cancer patient-derived cells (GC PDCs) and their respective xenograft tumors with different sensitivities to 5-fluorouracil (5-FU) by using immunodeficient female BALB/c-nu mice. RNA sequencing analysis of 5-FU-treated PDCs demonstrated that DNA replication/cell cycle-related genes were transiently induced in the earlier phase of DTP cell emergence, while extracellular matrix (ECM)-related genes were sustainably upregulated during long-term cell survival in 5-FU-resistant residual tumors. NicheNet analysis, which uncovers cell-cell signal interactions, indicated the transforming growth factor-ß (TGF-ß) pathway as the upstream regulator in response to 5-FU treatment. This induced ECM-related gene expression in the 5-FU-resistant tumor model. In the 5-FU-resistant residual tumors, there was a marked upregulation of cancer cell-derived TGF-ß1 expression and increased phosphorylation of SMAD3, a downstream regulator of the TGF-ß receptor. By contrast, these responses were not observed in a 5-FU-sensitive tumor model. We further found that TGF-ß-related upregulation of ECM genes was preferentially observed in non-responders to chemotherapy with 5-FU and/or oxaliplatin among 22 patient-derived xenograft tumors. These observations suggest that chemotherapy-induced activation of the TGF-ß1/SMAD3/ECM-related gene axis is a potential biomarker for the emergence of drug resistance in GCs.

Osteoblast responsive biosilica-enriched gelatin microfibrillar microenvironments.

Engineering of scaffolds for bone regeneration is often inspired by the native extracellular matrix mimicking its composite fibrous structure. In the present study, we used low loadings of diatomite earth (DE) biosilica to improve the bone regeneration potential of gelatin electrospun fibrillar microenvironments. We explored the effect of increasing the DE content from 1 % to 3 % and 5 %, respectively, on the physico-chemical properties of the fibrous scaffolds denoted FG_DE1, FG_DE3, FG_DE5, regarding the aqueous media affinity, stability under simulated physiological conditions, morphology characteristics, and local mechanical properties at the surface. The presence of biosilica generated composite structures with lower swelling degrees and higher stiffness when compared to gelatin fibers. Increasing DE content led to higher Young modulus, while the stability of the protein matrix in PBS, at 37 °C, over 21 was significantly decreased by the presence of diatomite loadings. The best preosteoblast response was obtained for FG_DE3, with enhanced mineralization during the osteogenic differentiation when compared to the control sample without diatomite. 5 % DE in FG_DE5 proved to negatively influence cells' metabolic activity and morphology. Hence, the obtained composite microfibrillar scaffolds might find application as osteoblast-responsive materials for bone tissue engineering.

Tubastatin alleviates epidural fibrosis via negatively targeting TGFß/PI3K/Akt pathway.

Epidural fibrosis (EF) is a chronic, progressive and severe disease. Histone deacetylase 6 (HDAC6) regulates biological signals and cell activities by deacetylating lysine residues and participates in TGF-ß-induced epithelial-mesenchymal transition (EMT). Nevertheless, the effect and mechanism of HDAC6 in EF remain unclear. To investigate the effect and mechanism of HDAC6 inhibition on repressing epidural fibrosis. HDAC6 expression and α-smooth muscle actin (α-SMA) in normal human tissue and human EF tissue were assessed by quantitative real-time PCR (qRT-PCR) and western blotting. Human fibroblasts were treated with TGF-ß ± HDAC6 inhibitors (Tubastatin) and fibrotic markers including collagen I, collagen III, α-SMA and fibronectin were assessed using western blotting. Then TGFß1 receptor (TGFß1-R), PI3K and Akt were analyzed using qRT-PCR and western blotting. Rats were undergone laminectomy± Tubastatin (intraperitoneally injection; daily for 7 days) and epidural scar extracellular matrix (ECM) expression was gauged using immunoblots. Increasing HDAC6 expression was associated with α-SMA enrichment. Tubastatin remarkably restrained TGF-ß-induced level of collagen and ECM deposition in human fibroblasts, and the discovery was accompanied by decreased PI3K and Akt phosphorylation. Moreover, Tubastatin also inhibited TGF-ß-mediated HIF-1α and VEGF expression. In the epidural fibrosis model, we found that Tubastatin weakened scar hyperplasia and collagen deposition, and effectively inhibited the process of epidural fibrosis. These results indicated that Tubastatin inhibited HDAC6 expression and decreased TGF-ß/ PI3K/ Akt pathway that promotes collagen and ECM deposition and VEGF release, leading reduction of myofibroblast activation. Hence, Tubastatin ameliorated epidural fibrosis development.

An endometrial biomimetic extracellular matrix (ECM) for enhanced endometrial regeneration using hyaluronic acid hydrogel containing recombinant human type III collagen.

Endometrial injury poses a significant challenge in tissue regeneration, with type III collagen (COL III) playing a pivotal role in maintaining endometrial integrity and facilitating repair. Our study explored the utility of recombinant human type III collagen (RHC) as an intervention for endometrial damage. To address the challenges associated with the inherent instability and rapid degradation of COL III in vivo, we developed an RHC-HA hydrogel by conjugating RHC with hyaluronic acid (HA), thus ensuring a more stable and sustained delivery. Our findings suggested that the RHC-HA hydrogel significantly promoted endometrial regeneration and restored fertility. The hydrogel facilitated prolonged retention of RHC in the uterus, leading to a substantial improvement in the repair process. The synergistic interaction between RHC and HA greatly enhances cell proliferation and adhesion, surpassing the efficacy of HA or RHC alone. Additionally, the RHC-HA hydrogel demonstrated notable anti-fibrotic effects, which are crucial for preventing abnormalities during endometrial healing. These findings suggested that the RHC-HA hydrogel presented a therapeutic strategy in the treatment of uterine endometrial injuries, which may improve female reproductive health.

Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis.

BACKGROUND: Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury, and finally leads to liver cirrhosis or even hepatocellular carcinoma. The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells (HSCs), which can transdifferentiate into myofibroblasts to produce an excess of the extracellular matrix (ECM). Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis. Therefore, activated hepatic stellate cells (aHSCs), the principal ECM producing cells in the injured liver, are a promising therapeutic target for the treatment of hepatic fibrosis. AIM: To explore the effect of taurine on aHSC proliferation and the mechanisms involved. METHODS: Human HSCs (LX-2) were randomly divided into five groups: Normal control group, platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) treated group, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 mmol/L, respectively) with PDGF-BB (20 ng/mL) treated group. Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs. Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species (ROS), malondialdehyde, glutathione, and iron concentration. Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62. RESULTS: Taurine promoted the death of aHSCs and reduced the deposition of the ECM. Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation, by decreasing autophagosome formation, downregulating LC3B and Beclin 1 protein expression, and upregulating p62 protein expression. Meanwhile, treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Furthermore, bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4, exhibiting the best average binding affinity of -20.99 kcal/mol. CONCLUSION: Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

Aflatoxin B1 inhibited the development of primary myoblasts of grass carp (Ctenopharyngodon idella) by degrading extracellular matrix.

According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 µM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 µM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 µM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 µM AFB1 (P < 0.05), respectively. Furthermore, 15 µM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 µM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 µM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway.