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Ferroptosis has emerged as a deliberate type of programmed cell death that manifests marked importance ubiquitously in health and diseases. However, after a decade of research, the mechanisms of ferroptosis execution remain unclear. Here we identify chloride ions (Cl-) as essential determinants of ferroptosis. Water homeostasis manipulated by extracellular solute concentration disrupts ferroptotic cell death. Hyperosmotic stress attenuates ferroptosis and endues cells with high lipid peroxidation. Analyses of a fluorescent chloride probe show that Cl-fluxes into the cytoplasm during ferroptosis, substantiating a role for Cl-to drive water flow. Depletion of extracellular chloride ions ([Cl-]o) from culture media congruously confers resistance to ferroptosis. The [Cl-]o-depleted ferroptotic cells fall into two populations: cells with low lipid peroxidation; cells with high lipid peroxidation but not cell swelling or cell rupture. Contrarily, solitary [Cl-]ooverload is sufficient to elicit ferroptosis without canonical ferroptosis inducers. Further experiments show that ferroptotic cells depolarize and [Cl-]ois positively correlated with this process. Membrane depolarization upregulates the level of lipid peroxidation, suggesting that membrane potential may be a universal mechanism governing ferroptosis. Together, our findings reveal that ferroptosis is determined by chloride ions.

Rheumatoid arthritis (RA) is a chronic, autoimmune and systemic inflammatory disease affecting 1% of the population worldwide. Immune suppression of the activity and progress of RA is vital to reduce the disability and mortality rate as well as improve the quality of life of RA patients. However, the immune molecular mechanism of RA has not been clarified yet. Our results indicated that exosomes derived from TNFα-stimulated RA fibroblast-like synoviocytes (RA-FLSs) suppressed chondrocyte proliferation and migration through modulating cartilage extracellular matrix (CECM) determining by MTS assay, cell cycle analysis, Transwell assay and Western blot (WB). Besides, RNA sequencing and verification by qRT-PCR revealed that exosomal long non-coding RNA (lncRNA) tumor necrosis factor-associated factor 1 (TRAF1)-4:1 derived from RA-FLSs treated with TNFα was a candidate lncRNA, which also inhibited chondrocyte proliferation and migration through degrading CECM. Moreover, RNA sequencing and bioinformatics analysis identified that C-X-C motif chemokine ligand 1 (CXCL1) was a target mRNA of miR-27a-3p while miR-27a-3p was a target miRNA of lnc-TRAF1-4:1 in chondrocytes. Mechanistically, lnc-TRAF1-4:1 upregulated CXCL1 expression through sponging miR-27a-3p as a competing endogenous RNA (ceRNA) in chondrocytes identifying by Dual-luciferase reporter gene assay. Summarily, exosomal lncRNA TRAFD1-4:1 derived from RA-FLSs suppressed chondrocyte proliferation and migration through degrading CECM by upregulating CXCL1 as a sponge of miR-27a-3p. This study uncovered a novel RA-related lncRNA and investigated the roles of RA-FLS-derived exosomes and exosomal lnc-TRAF1-4:1 in articular cartilage impairment, which might provide novel therapeutic targets for RA.

Failing to regenerate native tendon tissue in chronic massive rotator cuff tears (CMRCTs) results in high retear rates after surgery. Gelatin is a hydrolyzed form of collagen which is bioactive and biocompatible. This study intends to investigate the suitability of integrating gelatin to PLLA fibrous membranes for promoting the healing of CMRCTs. PLLA/Gelatin electrospun membranes (PGEM) are fabricated using electrospinning technology. The FTIR, static contact angles are tested sequentially. Cytocompatibility is evaluated with rat tendon fibroblasts and human umbilical endothelial cells (HUEVCs) lines. CMRCTs rat models are established and assigned into three groups (the sham group, the repaired group, and the augmentation group) to perform histomorphological and biomechanical evaluations. Gelatin is successfully integrated into PLLA fibrous membranes by the electrospinning technique. In vitro studies indicate that PGEM shows a great cytocompatibility for rat tendon fibroblasts and HUEVCs. In vivo studies find that applications of PGEM significantly promote well-aligned collagen I fibers formation and enhance biomechanical properties of the repaired tendon in CMRCTs rat models. In summary, gelatin promotes tendon fibroblasts and HUEVCs adhesion, migration, and proliferation on the PLLA fibrous membranes, and PGEM may provide a great prospect for clinical application. This article is protected by copyright. All rights reserved.

This research was set up to determine the efficacy of a synthetic biomimetic skin substitute of poly-L-lactide (PLLA)/gelatin nanofiber membrane in facilitating chronic cutaneous wound healing. The...

Objective Autograft microskin transplantation has been widely used as a skin graft therapy in full-thickness skin defect. However, skin grafting failure can lead to a pathological delay wound healing due to a poor vascularization bed. Considering the active role of adipose-derived stem cell (ADSC) in promoting angiogenesis, we intend to investigate the efficacy of autograft microskin combined with ADSC transplantation for facilitating wound healing in a full-thickness skin defect mouse model. Material and methods An in vivo full-thickness skin defect mouse model was used to evaluate the contribution of transplantation microskin and ADSC in wound healing. The angiogenesis was detected by immunohistochemistry staining. In vitro paracrine signaling pathway was evaluated by protein array and Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction network analysis. Results Co-transplantation of microskin and ADSC potentiated the wound healing with better epithelization, smaller scar thickness, and higher angiogenesis (CD31) in the subcutaneous layer. We found both EGF and VEGF cytokines were secreted by microskin in vitro. Additionally, secretome proteomic analysis in a co-culture system of microskin and ADSC revealed that ADSC could secrete a wide range of important molecules to form a reacting network with microskin, including VEGF, IL-6, EGF, uPAR, MCP-3, G-CSF, and Tie-2, which most likely supported the angiogenesis effect as observed. Conclusion Overall, we concluded that the use of ADSC partially modulates microskin function and enhances wound healing by promoting angiogenesis in a full-thickness skin defect mouse model. Electronic supplementary material The online version of this article (10.1186/s13287-019-1389-4) contains supplementary material, which is available to authorized users.

Joint contracture is a fibrotic complication induced by joint immobilization and trauma, which is characterized as excessive myofibroblast proliferation in joint capsule. The treatments of joint contracture are unsatisfied and patients are suffered from joint dysfunction. Our previous study has shown that curcumin can inhibit myofibroblast proliferation in vitro, but the major challenge is the low aqueous solubility and biological activity of curcumin. In this study, hyaluronic acid-curcumin (HA-Cur) conjugate was synthesized to suppress myofibroblasts in joint contracture. Cells were isolated from the joint capsules of joint contracture patients and induced to active myofibroblasts by transforming growth factor-β (TGF-β). The anti-fibrotic function and mechanisms of HA-Cur were investigated by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (PCR), methylation-specific PCR, western blot, transwell migration assay and proliferation assay. Results showed that 30 μM HA-Cur significantly attenuated the fibrotic functions of myofibroblast in joint contracture in vitro by regulating the methylation of prostaglandin E receptor 2 (PTGER2) and inhibiting TGF-β signaling. This may provide a mechanism for the treatment of joint contracture, and provide a molecular target PTGER2 for therapy during the pathogenesis of joint contracture.

Periosteum is clinically required for the management of large bone defects. Attempts to exploit the periosteum's participation in bone healing, however, have rarely featured biological and mechanical complexity for the scaffolds relevant to translational medicine. In this regard, we report engineering of bioinspired periosteum with co-delivery of ionic and geometry cues. The scaffold demonstrated microsheet-like fibre morphology and was developed based on bioresorbable poly(-caprolactone) and bioactive copper-doped tricalcium phosphate (Cu-TCP). A coordinated interaction was found between the effects of Cu-TCP addition and uniaxial drawing, leading to tunable fibrogenesis for different fibre morphologies, organisation, and surface wettability. The coordination resulted in significant enhancements in Young's Modulus, yield stress and ultimate stress along fibrous alignment, without causing reductions across fibres. This demonstrated mechanical anisotropy of the scaffold similar to natural periosteum, and seeding with mouse calvarial preosteoblasts, the scaffold supported cell alignment with deposition of CaP-like nodules and extracellular matrix. This work provides new insights on periosteum engineering with osteo-related composite fibres. The artificial periosteum can be used in clinical settings to facilitate repair of large bone defects.

Osteosarcoma is the most common bone sarcoma in adolescents. Decorin (DCN) has been proposed to be a new anti-osteosarcoma therapeutic strategy. Our previous study has loaded decorin on titanium (Ti) surface by polydopamine (DOPA) as an anchor to enhance osseointegration. In this study, we investigated the effect of decorin-coated Ti substrates (TI-DOPA-DCN) on the oncogenic potential of osteosarcoma cells SAOS-2. The substrates were placed in 24-well plates for cell culture. Cell viability was determined by Cell Counting Kit-8 (CCK8) assay. Apoptosis was evaluated by DAPI staining and Annexin V-FITC/PI double staining analysis. Cell cycle was analyzed by flow cytometry. Cell migration and invasion were evaluated by Transwell assay. For co-culture, the pre-osteogenic cells MEC3T3-E1 and osteosarcoma cells SAOS-2 were stained with cell membrane fluorescent dyes, and then mixed (1:1) for co-culture. The cells were observed under a fluorescence microscope at four time points of 24, 48, 72, and 96 h. The results showed that TI-DOPA-DCN substrate can selectively inhibit cell proliferation of osteosarcoma cells but not pre-osteoblasts. However, the cell cycle of SAOS-2 was not affected by TI-DOPA-DCN substrates. Both DAPI staining and Annexin V-FITC/PI double staining analysis revealed that TI-DOPA-DCN substrates induced apoptosis of osteosarcoma cells. Transwell assay showed that TI-DOPA-DCN substrates inhibited invasion and migration of osteosarcoma cells. Moreover, TI-DOPA-DCN substrates inhibited the growth of osteosarcoma cells but promoted that of pre-osteoblasts in the coculture system. Taken together, these findings suggested that decorin coating on Ti surface simultaneously inhibited the oncogenic potential of osteosarcoma cells but enhanced cell growth of pre-osteoblasts, which could be applied to surface modification of Ti orthopedic implant.

Osteosarcoma is a highly malignant bone cancer which primarily occurs in children and young adults. Increasing evidence indicates that long noncoding RNAs (lncRNAs), which function as competing endogenous RNAs (ceRNAs) that sponge microRNAs (miRNAs) and messenger RNAs (mRNAs), play a pivotal role in the pathogenesis and progression of cancers. The regulatory mechanisms of lncRNA-mediated ceRNAs in osteosarcoma have not been fully elucidated. In this study, we identified differentially expressed lncRNAs, miRNAs, and mRNAs in osteosarcoma based on RNA microarray profiles in the Gene Expression Omnibus (GEO) database. A ceRNA network was constructed utilizing bioinformatic tools. Kaplan-Meier survival analysis showed that lncR-C3orf35 and HMGB1 were associated with poor prognosis of osteosarcoma patients. Furthermore, results of Gene Set Enrichment Analysis (GSEA) suggested that lncR-C3orf35 may be involved in cellular invasion, the Toll-like receptor signaling pathway, and immune cell infiltration in the tumor microenvironment. Further analysis showed that patients with osteosarcoma metastasis expressed higher levels of lncR-C3orf35 and HMGB1 compared to metastasis-free patients. Moreover, the metastasis-free survival rate of the high lncR-C3orf35/HMGB1 expression group was significantly lower than that of the low expression group. The ESTIMATE algorithm was used to calculate the immune score and stromal scores for each sample. High lncR-C3orf35 and HMGB1 levels were correlated with low immune scores. ImmuCellAI analysis revealed that a low proportion of macrophage infiltration was associated with high lncR-C3orf35 and HMGB1 expression. The differential expression of lncR-C3orf35, miR-142-3p, and HMGB1 was further verified by quantitative real-time PCR. This study indicates that lncR-C3orf35 could be considered as a novel potential biomarker and therapeutic target of osteosarcoma, which may contribute to a better understanding of ceRNA regulatory mechanisms.

Joint contracture is a common complication for people with joint immobility that involves fibrosis structural alteration in the joint capsule. Considering that endoplasmic reticulum (ER) stress plays a prominent role in the promotion of tissue fibrosis, we investigated whether the unfolded protein response (UPR) contributes to the fibrotic development in immobilization-induced knee joint contractures. Using a non-traumatic rat knee joint contracture model, twelve female Sprague-Dawley rats received knee joint immobilization for a period of 8 weeks. We found that fibrosis protein markers (type I collagen, α-SMA) and UPR (GRP78, ATF6α, XBP1s) markers were parallelly upregulated in rat primary cultured synovial myofibroblasts. In the same cell types, pre-treatment with an ER stress inhibitor, 4-phenylbutyric acid (4-PBA), not only abrogated cytokine TGFβ1 stimulation but also reduced the protein level of UPR. Additionally, high reactive oxygen species (ROS) generation was detected in synovial myofibroblasts through flow cytometry, as expected. Notably, TGFβ1-induced UPR was significantly reduced through the inhibition of ROS with antioxidants. These data suggest that ER stress act as a pro-fibrotic stimulus through the overexpression of ROS in synovial fibroblasts. Interestingly, immunohistochemical results showed an increase in the UPR protein levels both in human acquired joint contractures capsule tissue and in animal knee joint contracture tissue. Together, our findings suggest that ER stress contributes to synovial myofibroblastic differentiation in joint capsule fibrosis and may also serve as a potential therapeutic target in joint contractures.

Hip arthroscopy is an effective method for the diagnosis and treatment of hip joint pathologies. However, gaining access to the central and peripheral compartments is challenging. The present study aimed to assess the advantages of using an arthroscopic extra-capsular approach and partial capsulotomy for access and subsequent management of hip diseases. Patients subjected to hip arthroscopy by partial capsulotomy for exposure and treatment of hip diseases between February 2012 and February 2016 were retrospectively analyzed. A total of 32 patients, including 19 males and 13 females, aged 19-48 years (median age, 36 years), had undergone the procedure. Firstly, the distal anterior lateral and anterolateral arthroscopic approach with blunt dissection was performed. Subsequently, a T-shaped partial capsulotomy was established to achieve adequate exposure. The shaver, radiofrequency probe and tissue penetrating suture grasper were then inserted to perform procedures including debridement of the synovium, suturing of the glenoid labrum. During surgery, a probe hook was used to push the capsule section limbs or pull the sutures placed on the capsule section limbs to improve exposure. For patients with pre-operative anterior instability, ligamentous laxity or acetabular dysplasia capsules were sutured to finish capsule closure. The pre-operative and post-operative Visual Analogue Scale (VAS) score and modified Harris hip score (MHHS) were used to assess the effectiveness of the procedure. No obvious post-operative complications were encountered. The mean follow-up time was 22.4 months (range, 18-32 months) and 31 patients completed the follow-up, while 1 patient was lost to follow-up. Compared with the pre-operative score, the MHHS was significantly increased (66.2±6.0 vs. 82.6±5.2; P

Orthopedic implants, using materials such as titanium, are extensively used in clinical surgeries. Despite its popularity, titanium is still inadequate to reliable osseointegration due to aseptic loosing. Fibrous encapsulation on the titanium implant interface prevents osseointegration and leads to the loosing of orthopedic implant. In this study, decorin was loaded on titanium surface by polydopamine film to examine fibrous encapsulation inhibition and bone growth acceleration. The coating of decorin was evaluated by X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. Quantitative analysis showed increased decorin coating on titanium surface when decorin in the loading solution increases. To test the effect of decorin modification, fibroblast and osteoblast cultures were utilized in vitro. The results showed that the functions of fibroblasts (proliferation, migration and collagen synthesis) were significantly attenuated on the decorin-modified surfaces and this anti-fibrous effect could be due to fibrotic gene suppression by decorin. In contrast, osteoblastic activities, such as calcium deposition and alkaline phosphatase (ALP) activity, were enhanced by the modified decorin. These results suggest that decorin coating on titanium surface inhibited proliferation and function of fibroblasts and improved that of osteoblasts. Therefore, this study is potentially useful for enhancing orthopedic implant.

Osteosarcoma (OS) is the most common primary solid malignant bone tumor, and its metastasis is a prominent cause of high mortality in patients. In this study, a prognosis risk signature was constructed based on metastasis-associated genes. Four microarrays datasets with clinical information were downloaded from Gene Expression Omnibus, and 256 metastasis-associated genes were identified by limma package. Further, a protein-protein interaction network was constructed, and survival analysis was performed using data from the Therapeutically Applicable Research to Generate Effective Treatments data matrix, identifying 19 genes correlated with prognosis. Six genes were selected by the least absolute shrinkage and selection operator regression for multivariate cox analysis. Finally, a three-gene (MYC, CPE, and LY86) risk signature was constructed, and datasets GSE21257 and GSE16091 were used to validate the prediction efficiency of the signature. The survival times of low- and high-risk groups were significantly different in the training set and validation set. Additionally, gene set enrichment analysis revealed that the genes in the signature may affect the cell cycle, gap junctions, and interleukin-6 production. Therefore, the three-gene survival risk signature could potentially predict the prognosis of patients with OS. Further, proteins encoded by CPE and LY86 may provide novel insights into the prediction of OS prognosis and therapeutic targets.

Joint contracture is increasingly regarded as a clinical problem that leads to irreversible dysfunction of the joint. It is a pathophysiological process following joint injury, which is marked by the activation of myofibroblasts. There is currently no effective treatment for the prevention of joint contracture. Curcumin is a polyphenol pigment extracted from turmeric, which possesses anti-inflammatory, antioxidative, and antitumor properties. In the present study, we demonstrated that curcumin exerts a protective effect against joint contracture via the inhibition of myofibroblast proliferation and migration in a time- and concentration-dependent manner. Moreover, we indicated that phosphatase and tension homolog (PTEN) was downregulated in myofibroblasts in vitro and in the contracture capsule tissues of patients in vivo. Additionally, western blot analysis revealed a negative correlation between the expression levels of PTEN and the fibrosis marker protein alpha smooth muscle cell actin. Methylation-specific PCR results suggested that curcumin was able to demethylate PTEN in a similar manner to the demethylation agent 5-azacytidine, increasing PTEN expression and further inhibiting phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling. In conclusion, our data illustrate part of the mechanism of curcumin inhibition in joint contracture. These results support the hypothesis that curcumin may potentially be used as a novel candidate for the treatment of joint contracture.

Table S1. Primers of quantitative reverse transcriptionâ polymerase chain reaction (qRT-PCR). (DOC 29 kb)

Table S1. List of siRNA sequences used in this study. Figure S1. Immunohistochemistry analysis of the expression of CCT6b in human contractive knee capsule (A) and normal control sample (B). Arrows indicate the CCT6b staining (magnification Ă â 10). (DOCX 2594 kb)

Background Joint contracture is a fibrous disease characterized as joint capsule fibrosis that results in joint dysfunction and disability. The purpose of this study was to analyze the biological activities of chaperonin containing T-complex polypeptide (CCT) subunits and to determine the role of CCT chaperone in joint contracture in a rat model. Methods In this study, the rat model of joint contracture was established by immobilizing the rat knee for 8 weeks. Then, fibroblasts were isolated from the posterior joint capsule and were cultured for functional analysis such as qRT-PCR, Western blot, transwell assay, and collagen assay. The effect of CCT subunit was determined by employing a lentivirus containing target gene and transfecting it into fibroblasts. Results Results of qRT-PCR and Western blot showed that among all CCT subunits, CCT6b significantly decreased in the fibroblasts from contractive joints compared to cells from normal joints (p < 0.05). Overexpression of CCT6b by transfection of lentivirus containing CCT6b gene to active fibroblasts significantly inhibited fibrous marker (α-SMA, COL-1) expressions, fibroblast migration, and collagen synthesis (all p < 0.05). Moreover, fibrosis-related chaperone CCT7 expression was decreased with CCT6b overexpression (p < 0.05). Conclusion The biological activities of CCT subunits in fibroblasts from the joint contracture rat model were analyzed in this study. CCT6b significantly decreased in the active fibroblasts, and overexpression of CCT6b significantly inhibited fibroblast functions. These findings indicate that CCT6b appears to be a potential molecular biomarker and therapeutic target for the novel therapies of joint contracture. Electronic supplementary material The online version of this article (10.1186/s13018-019-1161-6) contains supplementary material, which is available to authorized users.

Joint contracture is a fibroproliferative disorder that restricts joint mobility, resulting in tissue degeneration and deformity. However, the etiology of joint contracture is still unknown. Chaperonin containing T-complex polypeptide subunit eta (CCT-eta) is reported to increase in fibrotic diseases. The purpose of this study was to investigate whether CCT-eta is implicated in joint contracture and to determine the role of CCT-eta in the progression of joint contracture by analyzing a rat model. We immobilized the left knee joint of rat by internal fixation for 8 weeks. The non-immobilized right leg served as a control. The range of motion (ROM) of the knee was investigated. Fibroblasts were obtained from the posterior joint capsule of the joints. The outcome was followed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, fibroblast migration assay, and collagen assay. The effect of CCT-eta on the functions of fibroblasts was observed by utilizing a short inhibitory RNA (siRNA) targeting CCT-eta. The ROM of the immobilized joints was significantly limited compared to the contralateral joints (p

Fibrous encapsulation that prevents the direct contact between an implant and the bone can cause implant failure. However, prevention of fibrous encapsulation is difficult because of the lack of effective strategies which can selectively control the growth of fibroblasts and osteoblasts. Because curcumin, an extract from Curcuma longa, was recently found to reduce the formation of fibrous tissue, it is hypothesized that loading curcumin on implant surfaces would be efficacious in inhibiting fibrous encapsulation without adversely affecting the osteoblast functions. To prove this hypothesis, curcumin was loaded on to a titanium surface using poly(dopamine) as an anchor, and the behaviors of fibroblasts and osteoblasts on these curcumin-modified surfaces were investigated. Curcumin was successfully loaded on to titanium and showed a low release after incubation in phosphate-buffered saline for seven days. On the curcumin-modified surfaces, fibroblast proliferation was suppressed, and fibrous marker expressions as well as collagen synthesis were significantly reduced. These reductions were possibly because of the enhancement of fibroblast apoptosis induced by the surface curcumin. In contrast, no significant reduction in osteoblast functions was observed on the curcumin-modified substrates. These findings may provide a promising solution to reduce fibrous encapsulation, and thus may be highly beneficial for orthopaedic applications.