Osteoarthritis (OA) is a degenerative joint disorder commonly encountered in clinical practice, and is the leading cause of disability in elderly people. Due to the poor self-healing capacity of articular cartilage and lack of specific diagnostic biomarkers, OA is a challenging disease with limited treatment options. Traditional pharmacologic therapies such as acetaminophen, non-steroidal anti-inflammatory drugs, and opioids are effective in relieving pain but are incapable of reversing cartilage damage and are frequently associated with adverse events. Current research focuses on the development of new OA drugs , which aims for more effectiveness and less incidence of adverse effects than the traditional ones. Furthermore, regenerative therapies (such as autologous chondrocyte implantation (ACI), new generation of matrix-induced ACI, cell-free scaffolds, induced pluripotent stem cells (iPS cells or iPSCs), and endogenous cell homing) are also emerging as promising alternatives as they have potential to enhance cartilage repair, and ultimately restore healthy tissue. However, despite currently available therapies and research advances, there remain unmet medical needs in the treatment of OA. This review highlights current research progress on pharmacologic and regenerative therapies for OA including key advances and potential limitations. The symptoms of OA include joint pain, swelling, tenderness, stiffness, and sometimes locking, which may lead to disability and severely affect the life quality of patients.Osteoarthritis (OA), also known as degenerative joint disease, is characterized by cartilage degeneration and osseous overgrowth. OA is commonly encountered in today’s clinical practice. The incidence of OA increases with age, and it is one of the most prevalent diseases in older people. In the USA alone, 10% of men and 13% of women aged 60 and older have been diagnosed with knee OA. Normally, applied pharmacologic therapy shows efficacy in pain relief but is frequently associated with adverse events. In recent years, emerging regenerative therapy has gained much attention as it can efficiently promote tissue repair and regeneration.Due to the lack of self-healing capacity of articular cartilage, OA is among the most challenging joint diseases and there is currently no cure for it. The focus of treatment for OA is to reduce pain and improve function of the affected joints.This review mainly focuses on the current pharmacologic and regenerative therapeutic approaches for OA treatment. It includes therapy that has been confirmed clinically effective and used for decades, as well as therapy that shows promise in preclinical research and can potentially be translated for future clinical application, offering a systematic overview of progress in OA treatment in relation to progress with pharmacologic and regenerative therapy. which transmit loads, stabilize the matrix, and maintain a healthy cartilage microenvironment. Due to its load-bearing function, cartilage is highly susceptible to damage during sports activities, and wear and tear over time. First proposed by Hunter in 1742, it has long been recognized that cartilage defects cannot heal spontaneously. Researchers believe that the poor self-healing capacity is probably due to the poor blood supply and low metabolic activity in cartilage. If injured cartilage is not treated, it gets worse and affects surrounding tissue, and ultimately degenerates into OA.Articular cartilage is a typical hyaline cartilage that covers the ends of bones making up the joints in the body. It maintains smooth and frictionless movement, and dissipates stress in the joint. Articular cartilage is an avascular and aneural tissue; it consists primarily of chondrocytes and extracellular matrix including collagen type 2 and proteoglycans, thus perturbing the extracellular matrix structure and impairing the biomechanical properties. Chondrocytes near the superficial layer form clusters, whereas in the deep and calcified layers, they undergo apoptotic death. Chondrocyte proliferation is somewhat activated, but cannot resist the predominant catabolic activity. During disease progression, OA chondrocytes produce matrix-degrading enzymes including matrix metalloproteinase 13 (MMP13), which degrades collagen and A disintegrin, and metalloproteinase with thrombospondin motifs-5 (Adamts-5), which targets aggrecan. The synthesis of degradative enzymes further exacerbates the breakdown of articular cartilage. The biomechanical and biochemical changes would together disrupt cartilage homeostasis and contribute to the pathogenesis of OA, which leads to joint space narrowing, painful cartilage destruction, and loss of function.Microscopically, OA cartilage is characterized by loss of collagen and proteoglycans, Osseous outgrowths called osteophytes often form at the joint margins. Subchondral bone sclerosis, meniscal tear and extrusion, and synovial membrane inflammation (synovitis) may also occur due to the mechanical changes in OA cartilage, and make OA disease more debilitating . In addition, the long-term results of arthroplasty differ significantly.37According to the Osteoarthritis Research Society International (OARSI) and the American Academy of Orthopaedic Surgeons (AAOS), the mainstay of OA treatments involves physical measures, drug therapy, and surgery.38 It has already been applied in the orthopaedic clinic for several decades and promising outcomes have been achieved.39 In recent years, pilot clinical studies, although few, show the usefulness of regenerative therapy in the treatment of OA, suggesting its potential to be translated from bench to bedside.40–47 In the next sections, we would like to focus on the current pharmaceutical and regenerative therapies that have been clinically investigated, and that show safety and effectiveness in the management of OA.Pharmaceutical therapy is the most commonly used OA treatment option aimed mainly at pain relief and anti-inflammation. The traditional OA drugs are limited to control OA symptoms, but none can reverse the damage in the OA joint. And, the traditional drugs are always overwhelmed by its high incidence of adverse effects. Studies of new OA drugs (mainly biologic agents) with more effectiveness and fewer side effects are underway. In addition, regenerative therapy holds the possibility of repairing and regenerating damaged or lost tissues to restore the original structure and function.There are mainly five kinds of medications commonly used in today’s clinical treatment of OA: acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, serotonin–norepinephrine reuptake inhibitors (SNRIs), and intra-articular injections. As there has been a considerable amount of literature focusing on the traditional drugs, here we provide a brief updated overview. The recommendations of the AAOS, American College of Rheumatology (ACR) and OARSI are summarized in 49 Overdosing acetaminophen may be toxic to the liver.51 Due to the risk of liver damage, on 13 January 2011, the US Food and Drug Administration (FDA) limited the amount of acetaminophen in prescription combination products to no >325 mg per dosage unit.52 Consistent with the change made by the FDA, the latest 2013 AAOS guideline downgraded the acetaminophen recommendation level to inconclusive and reduced the daily dosage from 4 000 to 3 000 mg.24 For patients with severe symptoms or who do not respond to acetaminophen, more potent drugs should be considered, such as NSAIDs.Acetaminophen or paracetamol is an essential medicine as recognized by the World Health Organization, and is commonly used to reduce fever and relieve pains such as headache, muscle aches, backaches, and toothaches. Due to its relative safety and effectiveness, acetaminophen is recommended as the first-line oral analgesic for mild-to-moderate OA by most guidelines. According to ACR and OARSI guidelines, up to 4000 mg per day is an effective initial treatment for mild-to-moderate knee or hip OA.54 By analyzing eight randomized controlled trials (RCTs), NSAIDs were better overall than acetaminophen in terms of pain relief. Although the efficacy of NSAIDs for OA treatment has been well documented, the health concerns, however, greatly restrict their extensive application. It is estimated that the occurrence of adverse effects is ~30% in people taking NSAIDs.55 A total of 1%–2% of people using NSAIDs develop gastrointestinal (GI) complications per year, which is much higher than that of people who do not use NSAIDs.57 Although selective COX-2 inhibitors appeared safer than traditional NSAIDs, several commercial drugs have been placed under scrutiny or withdrawn by the FDA. The first approved COX-2 inhibitor Celecoxib  received an FDA alert for the potential risk of serious adverse cardiovascular events.58 Rofecoxib  and Valdecoxib  were withdrawn from the market for associated cardiovascular risks and other side effects.60 Therefore, there is a balance between the efficacy and safety of NSAIDs, and the benefit/risk ratio should be considered when taking these drugs. It is recommended by OARSI that NSAIDs be used at the minimum effective dose and prolonged use should be avoided as much as possible.3NSAIDs provide anti-inflammatory and analgesic effects, and have long been used as an important remedy for moderate-to-severe OA. Acetaminophen is not regarded as an NSAID as it has little anti-inflammatory effect. Some studies adopted meta-analysis to compare the safety and efficacy between acetaminophen and NSAIDs.3 There has been an increased use of opioids in OA treatment (31% opioid prescribed in 2003 to 40% in 2009),61 however, the frequent adverse effects associated with opioids, including nausea, vomiting, dizziness, constipation, sleepiness, tiredness, and headache, may outweigh the benefits in pain relief.62–64 Opioid abuse is another potential risk of using these drugs. Routine use should be avoided, and low effective and tolerated doses are recommended.Opioids are used for the management of moderate-to-severe pain when NSAIDs and acetaminophen are ineffective or contraindicated.65 Duloxetine was considered an acceptable and favorable OA treatment based on the results from two double-blind, placebo-controlled RCTs.67 It may be a promising and efficacious way to alleviate OA pain for patients who are unable to take other commonly used drugs. The FDA approval and ACR recommendation49 also confirm its value in clinical use. However, AAOS and OARSI have not included duloxetine in their OA management guidelines,24 as more large-scale longitudinal studies to further investigate the safety and efficacy for OA treatment have to be performed.SNRIs are primarily used in the treatment of depression and other mood disorders. In 2010, the FDA approved duloxetine, a selective SNRI, for the management of chronic musculoskeletal pain including OA.3 ACR and AAOS conditionally recommended corticosteroids for knee and/or for hip OA.49 Hyaluronic acid is a component in both healthy and OA joint fluid. Intra-articular injection of hyaluronic acid is recommended by OARIS as a treatment option for knee or hip OA.3 However, the efficacy of hyaluronic acid injection varies. The 2013 edition of the AAOS guideline downgraded the recommendation on hyaluronic acid from an inconclusive level to a non-affirming level after excluding the evidence of lower strength.24Intra-articular injection of corticosteroids and hyaluronic acid are selectively used in the treatment of OA. Corticosteroid injection is recommended by OARIS for patients with moderate-to-severe pain who do not respond to oral analgesic and anti-inflammatory agents.68The unsatisfactory effects and unacceptable side effects associated with traditional OA drugs warrant a continued search for potential new medications. Although few of them have received the regulatory approval for routine clinical use, a variety of new OA drugs have shown promising results in clinical trials . On the 69 A phase 1 safety and tolerability study first reported the use of BMP-7 in symptomatic knee OA.70 Thirty-three OA patients (mean age 60 years) were intra-articularly injected with four doses of BMP-7 or placebo. Participants who received 0.1 and 0.3 mg of BMP-7 showed greater symptomatic improvement and higher OARSI response rate. No dose-limiting toxicity was found. Phase 2 study with 0.1 and 0.3 mg dosing cohorts would be further conducted in future.Recombinant human bone morphogenetic protein-7 (BMP-7), also called osteogenic protein-1 (OP-1), was a FDA-approved biologic for the treatment of bone nonunions and spine fusion.71 and the other injected AMG108, a IL-1β receptor antibody, subcutaneously and intravenously in 159 patients.72 Although IL-1β receptor antagonist/antibody was well tolerated, no significant clinical improvements were reported compared with placebo in either study.Two randomized, double-blind, placebo-controlled studies attempted interleukin (IL)-1β inhibitor for knee OA treatment. One study administered IL-1β receptor antagonist intra-articularly in 160 patients,73 As compared with the placebo treatment, treatment with tanezumab significantly reduced knee pain while walking and improved the patients’ global assessment. However, 68% of patients receiving tanezumab were recorded with adverse events. Sixteen subjects developed rapidly progressive OA and required total joint replacements, prompting the FDA to request the suspension of the trials of tanezumab. However, from subsequent assessments, the risk of rapidly progressive OA with tanezumab was lower than that with tanezumab/NSAID combination therapy, and the rate of joint replacement was comparable between tanezumab monotherapy and placebo treatment.75 Therefore, the FDA has agreed to continue the clinical trials of tanezumab in OA treatment in conjunction with appropriate safety monitoring.Tanezumab, a monoclonal antibody against β-nerve growth factor, has been tested clinically against OA. A proof-of-concept study of tanezumab was performed in 450 patients with knee OA.76 More basic and clinical studies should be performed to fully investigate this novel OA biologic drug.The ideal biologic agents for OA treatment should alleviate pain, relieve symptoms and restore the normal structure of the joint. To date, no structure-modifying treatment has yet been approved. A proof-of-concept study has been conducted to evaluate the efficacy and safety of intraarticular sprifermin (recombinant human fibroblast growth factor-18) to treat symptomatic knee OA with 180 patients. Sprifermin treatment significantly reduced the loss of total and lateral femorotibial cartilage thickness and volume, as well as the joint space width narrowing in the lateral femorotibial compartment in a dose-dependent manner. No significant difference in serious adverse events was recorded between groups.77 Wang-Saegusa et al.78 treated 312 OA patients with total three intra-articular injections of autologous plasma rich in growth factors. After 6 months, statistically significant differences were seen in the following assessment instruments: visual analogue scale (VAS), SF-36, the Western Ontario and McMaster Universities Arthritis Index (WOMAC), and Lequesne Index. No adverse effects were observed. Positive trends and safety profile of PRP were also reported in other studies,80 suggesting a feasible and potential treatment for OA.Relatively more studies have been reported regarding platelet-rich plasma (PRP), which contains several kinds of growth factors including transforming growth factor β1 (TGF-β1), platelet-derived growth factor, vascular endothelial growth factor, insulin-like growth factor-1, and hepatocyte growth factor.82 In 2014, Ampio Pharmaceuticals completed the phase 3 clinical trial for Ampion and achieved ⩾40% improvement in WOMAC pain and function compared with placebo controls at 20 weeks.83 A subsequent multiple injection clinical study for severe knee OA was completed in 2015 and assured that multiple injections of Ampion were safe and effective,84 showing much potential for future treatment of OA.Ampion, a <5 kDa ultrafiltrate of human serum albumin, is currently being developed by Ampio Pharmaceuticals  as an intra-articular injection to treat knee OA through suppressing pro-inflammatory cytokine production in T cells.85 Thirteen of 30 (43%) participants achieved ⩾30% reduction in VAS pain score and OARSI responder criteria. A pragmatic phase 3 RCT with anticipated 160 participants was also conducted by this group from 2014 to 2015.87 Although the study was completed, the actual efficacy has not yet been determined, as no published data were provided from this study.87 Another Egyptian group performed a randomized placebo-controlled trial with 144 patients to assess the efficacy of methotrexate in the treatment of symptomatic knee OA.88 After 28-week treatment, patients who received oral methotrexate showed significantly improvement in reducing pain and clinical synovitis compared with the placebo-treated group, indicating the dual benefit of methotrexate as a novel OA therapeutic option.Methotrexate, a chemotherapeutic drug normally used to treat rheumatoid arthritis, is being tested in OA treatment. The first open-label pilot study with 30 patients treated with oral methotrexate was conducted in 2011 and indicated an analgesic efficacy for methotrexate in knee OA.89 It offers a long-term solution to repair and regenerate cartilage, alleviate symptoms and finally delay OA progression. Currently, cell therapy is applicable to both mature cells and stem cells.From the 1980s, cell-based therapy has been applied for cartilage repair and has rapidly developed over the past 30 years.et al.,90 autologous chondrocyte implantation/transplantation (ACI/ACT) is widely used in clinical practice and more than 15 000 patients have received this treatment worldwide.91 ACI mainly includes three key steps.92 First, a small mass of cartilage tissue (~150–300 mg) is collected from a healthy and less weight-bearing area during an arthroscopic biopsy procedure. Then, the extracellular matrix is enzymatically removed, and chondrocytes are isolated and cultured in vitro to acquire enough cells to reimplant. Finally, chondrocytes are implanted into the damaged area of the articular cartilage in a second open-knee procedure. On the basis of the various implantation methods, three generations of ACI have been developed in the past 20 years.92 The first generation adopts a piece of periosteum sutured over the prepared defect which is taken from the patient’s tibia. Then, the chondrocyte suspension is injected under the periosteum, where it forms a bioreactive chamber to allow cell growth and maturation.90 The limitations of the first generation lie in the periosteal delamination and hypertrophy, which lead to the development of the second generation of ACI using a bilayer collagen membrane. This biomaterial-based membrane is also sutured over the defect and followed by cell suspension injected underneath. The advance in tissue engineering contributes tangibly to the third generation. Cultured chondrocytes are pre-seeded on a three-dimensional scaffold and trimmed to fit the defect size. The ‘all-in-one’ composite is then implanted to the defect area with the fixation of fibrin glue. No periosteum or sutures are used in this method. Therefore, the third generation of ACI is also called matrix-induced autologous chondrocyte implantation/transplantation (MACI/MACT). MACI shows evident benefits over classic ACI as it reduces the surgical time, minimizes the fixation invasion and ensures even and long-term cell maintenance. More details about the scaffolds used in MACI will be discussed in the section ‘tissue engineering’.First described by Brittberg et al.90 first performed classic ACI in 23 patients with full-thickness cartilage defects. Eighty-eight percent of patients with femoral condylar defects showed good or excellent results after 2-year transplantation, but only 28.6% of patients with patella injuries had a satisfying outcome at the 3-year time point. Postoperative arthroscopy revealed 11 out of the 15 biopsies showing hyaline-like cartilage. Long-term follow-ups were subsequently reported by the same group for up to 10 years.94 Treatment for isolated femoral condyle defects and osteochondritis dissecans achieved ~90% good to excellent results. In addition, groups of patients with multiple and patella lesions demonstrated >65% good outcomes. Adverse effects were reported in 52 out of 101 patients.94 The most common complications were periosteal hypertrophy (26 patients) and intraarticular adhesions (10 patients). Overall, ACI can be regarded as a reasonable treatment for deep cartilage defects.The clinical outcomes of ACI have been well documented in full-thickness and osteochondral defect repair. In 1994, Brittberg 95 However, cartilage damage with generalized OA was an exclusion criterion for treatment.96 This is because ACI is applicable to localized cartilage defects surrounded by healthy cartilage. OA cartilage, however, often affects the adjacent areas and disturbs the homeostasis of the whole joint cavity. In this degenerative microenvironment, the implanted chondrocytes may undergo undesired dedifferentiation or apoptosis,98 therefore undermining efficacy. In recent years, two trials tested the first-generation ACI for the more challenging OA disease.100 Minas et al.100 performed classic ACI on 153 patients with early OA changes with a mean age of 37.3 and average defect size of 4.9 cm2. At an average 5-year follow-up, treatment failure occurred in 12 knees that changed to joint arthroplasty. Among the patients considered without treatment failures, 92% experienced functional improvements, especially in the WOMAC pain and function scores. They concluded that ACI provided a plausible treatment for young OA patients and delayed the need for knee arthroplasty. Rosenberger et al. reported a case series of 56 patients older than 45 years, among which 32 patients were diagnosed with early degenerative changes (mean defect size 11.7 cm2).99 At the latest available follow-up, 72% of patients of all defect categories and 81% of OA patients experienced good or excellent clinical improvements. Three out of 32 OA patients were considered treatment failures. Their favorable findings pointed to consider classic ACI as a therapy for older age groups.The commercial product Carticel  was initially approved by the FDA in 1997 for the repair of symptomatic cartilage defects of the femoral condyle caused by acute or repetitive trauma.in vitro chondrocyte dedifferentiation, and donor-site morbidity caused by cartilage harvest.102 Mesenchymal stem cells (MSCs) are considered a potential cell source since they can be easily collected from various tissues such as bone marrow, adipose tissue, synovial membrane, and others, and have a high proliferation rate, chondro-differentiation capacity, and immunosuppressive activities.103–105Although no severe clinical safety issues have been associated with the ACI technique, there are still some problems including the limited cells available, multiple surgical procedures involved, et al.40 published a preliminary report of four patients with moderate-to-severe knee OA. Autologous BM-MSCs were cultured for 4–5 weeks, and 8×106–9×106 cells were injected into the knee joint. After 1-year follow-up, pain produced during walking was reduced in three patients. The number of stairs to climb to produce pain and pain on a VAS were improved in all four patients. As the physical parameters improved slightly, the results were encouraging, but not ideal. In another trail, Orozco et al.41 performed MSC therapy on 12 patients diagnosed with Kellgren and Lawrence grades II to IV knee OA. More BM-MSCs (40×106) were intra-articularly injected. One-year follow-up indicated marked increase in VAS (69%), Lequesne (65%), and WOMAC (78%) pain indices. Cartilage quality was significantly improved in 11 of 12 patients as evidenced by T2 mapping quantification.Bone marrow-derived MSCs (BM-MSCs) are the most attractive stem cells in regenerative medicine studies, and attempts have been made to use them for OA treatment. In 2011, Davatchi 106 They proposed that AD-MSCs had an advantage over BM-MSCs as obtaining cells from bone marrow is difficult and painful, together with risks of complications. They collected the cells from the infrapatellar fat pad and prepared these with PRP. Twenty-five patients with knee OA received this intra-articular injection. It yielded improved clinical outcomes on the 1-year follow-up as shown by the Lysholm, Tegner activity scale, and VAS scores; but no significant difference was detected between the MSC-treated group and the control group, which consisted of injections of PRP alone.106 On 2-year follow-up to their previous study,42 WOMAC, Lysholm, and VAS pain scores were as well significantly improved when compared with the preoperative status. Magnetic resonance imaging (MRI) examination further confirmed the improvement in cartilage. However, no comparison between the treatment and control groups was shown. Thus, one cannot distinguish the effect of AD-MSCs from that of PRP and accurately investigate the efficacy of AD-MSCs on OA treatment.Choi’s group tried to use adipose tissue-derived MSCs (AD-MSCs) to treat OA.Overall, the preliminary results demonstrated that MSC-based therapy is encouraging in reducing pain and improving the function of OA. More RCTs with a large number of patients and long-term follow-up are needed before full-scale clinical translation.108 For OA treatment, only a few results have been reported, though there is hope for the future.Tissue engineering involves the use of cells, scaffolds, and bioactive factors to enhance tissue mechanical properties and promote cell migration, attachment, proliferation, and differentiation to the desired cell type. Tissue engineering therapy has shown a lot of promising outcomes in the treatment of cartilage defects.et al.,43 18 young patients suffering from medial knee OA (mean age 47 years) underwent high tibial osteotomy (HTO) combined with MACI using a collagen membrane scaffold . At the 5-year follow-up, the Knee Injury and Osteoarthritis Outcome Score (KOOS) was significantly improved. MRI results were improved at 24 and 48 months, but declined at the end point with only 33% good quality infill. No major complications but minor complications were found including patellar tendinitis. In another clinical trial, 79 patients with posttraumatic and focal OA cartilage defects were treated with autologous chondrocyte-seeded BioSeed-C scaffold. Clinical assessment was performed in 40 patients with 2-year follow-ups.44 The evaluated scores including International Knee Documentation Committee (IKDC) score, the Lysholm score, the Cincinnati knee score, and KOOS were all statistically significantly improved compared with preoperative values. Histological results showed good integration of the graft and newly formed cartilaginous tissue. In their subsequent 4-year follow-up with 19 patients of the cohort,45 the Lysholm score, IKDC score, and KOOS were further improved. MRI analysis revealed that 16 out of 19 patients experienced moderate to complete filling of the defects. These results showed that BioSeed-C is a potential therapeutic option for degenerative defects with stable effect. Although MACI technique has been reported with promising results for OA treatment in many trails, researchers demonstrated that MACI with Hyaff-11 scaffold was questionable for knee OA due to the poor performance and high failure rate. They treated 44 patients using MACI as a salvage procedure. After a 9-year mean follow-up, 27.3% treatment was considered to have failed. Almost half (47.7%) of the patients considered their condition unimproved and 39% would not choose this treatment again despite the significant improvement of IKDC and EQ-VAS scores. This long-term follow-up study indicated that the tissue-engineered cartilage implantation should be fully investigated before its application as a salvage procedure for the treatment of OA.Treatment with cell-based scaffolds involves tissue harvest and cell expansion procedures that are used in ACI or other forms of cell therapy. The cells are pre-seeded on the scaffold, and the composite is subsequently implanted into the defect area with or without fixation. The third-generation ACI (MACI) is one of the most extensively used techniques for the clinical treatment of cartilage defects. Many commercial products have been approved for scaffold-associated chondrocyte implantation for more than a decade in Europe and Australia, such as Chondro-Gide , Hyaff-11 , and BioSeed-C . In a case series published by Bauer 98 As exogenous cell transplantation is not required, it avoids the issues around the in vitro cell culture process, such as slow growth and aberrant differentiation.102 Clinical results of cell-free scaffolds on OA treatment are few. A case report documented a 46-year-old athletic patient with International Cartilage Repair Society (ICRS) grade IV degenerative chondral lesions treated with a three-layer nanostructured biomimetic scaffold  together with HTO.46 At 1-year follow-up, the patient was pain-free and returned to a satisfactory functional level. MRI analysis showed hyaline-like articular cartilage and non-visible subchondral oedema. An Italian group reported clinical improvements using PRP-enriched polyglycolic acid (PGA)-hyaluronan scaffold .47 Fifty-two patients suffering degenerative chondral defects were treated, among which, 47 patients had grade I–III OA. The KOOS score was significantly increased, and histological staining revealed hyaline-like cartilage repair tissue at 1-year follow-up. The above two pilot studies confirmed the usefulness of cell-free scaffolds. However, an in vivo study using sheep OA model demonstrated that cell-free approaches were inferior to MACI by macroscopic and histological examinations.109 It remains to be seen whether cell-free scaffolds have more advantages over cell-seeded scaffolds in human studies.Cell-free scaffolds are developed for one-stage procedure techniques, which can be either implanted alone to attract the endogenous cells or combined with biological products such as concentrated bone marrow or PRP.110 This technique called TissueGene-C uses the retrovirally transduced allogeneic human chondrocytes overexpressing TGF-β1. Phase 1 and 2 trials have commenced, though results have not been published yet.112 From the published results of phase 1 study with 12 advanced OA patients,113 only some minor injection site reactions but no serious adverse events were observed after 1 year post dosing. Knee evaluation scores showed a dose-dependent improvement of symptoms. Phase 2 data, only available in abstract form,114 suggested a significant improvement in IKDC, WOMAC, and VAS scores without severe adverse events after 6 months. The placebo-controlled, double-blind, randomized phase 3 study was just completed on August 2015 but no study results have been posted as yet.115 The above-mentioned regenerative therapies are briefly summarized in Gene therapy enables the spatiotemporal control and persistent synthesis of gene products at target sites. Several preclinical studies have confirmed its safety and efficacy, and implicated its prospects, but few clinical trials have been conducted and no gene products have been approved for OA treatment. At present, only TGF-β gene therapy has been clinically investigated in USA and Korea.In this review, we presented the current progress of pharmacologic and regenerative therapy for OA treatment. The traditional OA drugs are effective in reducing pain and inflammation but insufficient to slow, stop, or reverse the joint damage, and are frequently associated with adverse effects. New OA drugs such as biologic agents and chemotherapeutic drugs show more marked effects and fewer side effects, and look more promising than traditional OA drugs. Regenerative therapy is a novel strategy that has the potential to restore normal structure and function of damaged cartilage. At present, clinical studies in regenerative therapy are in its infancy with relatively rare and low-level evidence of success. Larger, random, controlled, and long-term follow-up studies are expected to take place in the coming years to confirm its safety and effectiveness. Although current pharmacologic and regenerative therapy show great promises, limitations still exist. Potential therapies may be developed by exploring more therapeutic targets and methods. The emerging targets that have been confirmed in preclinical animal studies are also summarized in 117 Wang et al.118 intraperitoneally injected CL82198, the MMP13 inhibitor in a murine model of injury-induced knee OA, which effectively decelerated OA progression, increased extracellular matrix production, and inhibited chondrocyte apoptosis. In another study performed by Chen et al.,119 Adamts-5 inhibitor (114810) and hyaluronic acid hydrogel were combined to treat rat OA knee joints and significantly prevented the progression of cartilage degeneration. In addition, Syndecan-4 was identified to control the activation of Adamts-5; therefore, the application of Syndecan-4-specific antibody could prevent proteoglycan loss and cartilage breakdown in a mouse OA model.120 However, the only clinical study with MMP inhibitor (PG-116800) for OA treatment resulted in termination due to musculoskeletal toxicity without clear benefit, suggesting more preclinical studies are needed to fully assess the safety and effectiveness of those matrix degradation inhibitors, and devise ways to improve efficacy.121As mentioned above, MMP13 and Adamts-5 are the main matrix-degrading enzymes that play a key role in the development of OA. In recent studies, MMP13 and Adamts-5 have been identified as downstream target genes involved in both β-catenin and TGF-β signaling pathways during OA development.123 It has been well identified that parathyroid hormone-related protein (PTHrP) acts in conjunction with Indian hedgehog to inhibit chondrocyte hypertrophy and regulates endochondral ossification through a negative-feedback loop.125 A recent study showed that systematic administration of recombinant human PTH , the homolog of PTHrP could effectively inhibit cartilage degeneration and aberrant chondrocyte maturation in a surgically induced mouse OA model.126 In our own study, we found that intra-articular injection of recombinant human PTHrP (1–40) at 4–6 weeks post injury together with the implantation of collagen-silk scaffold significantly suppress chondrocyte terminal differentiation and promote chondrogenesis, therefore improving cartilage repair and regeneration in a rabbit osteochondral defect model.123 It is also reported that the inhibition of the hedgehog signaling could block the formation of hypertrophic chondrocytes and ameliorate OA development using small molecular inhibitors.128Current OA treatments aim to regenerate hyaline-like cartilage tissue. However, the repair tissue is often accompanied with undesirable chondrocyte hypertrophy and terminal differentiation, which cause matrix degradation and then impair the function of the repair tissue.129 The Wnt/β-catenin signaling pathway has been demonstrated to be involved in both cartilage and bone development.130 Overexpression of dickkopf-related protein-1, one of the Wnt antagonists, ameliorated the severity of OA in mice by inactivation of the Wnt/β-catenin signaling in subchondral bone.131 These results not only suggested a potential treatment approach for OA disease but also shifted the treatment target from cartilage surface to subchondral bone, considering that OA is a disease of the whole joint.The therapeutic targets of most of today’s OA research are the articular cartilage itself, it is worthwhile to include the search of novel targets in the subchondral bone, which markedly becomes thicker and disrupted the mechanical stability in OA joints. Inhibition of TGF-β activity in subchondral bone may hold promise for OA treatment. Cao and colleagues reported in 2013 that injection of TGF-β type I receptor inhibitor (SB-505124) or the implantation of an antibody to TGF-β (1D11) in alginate beads could attenuate disease in ACLT-induced OA mice/rat.132 offering an ideal cell source for cartilage repair and OA treatment compared with adult chondrocytes or MSCs. Embryonic stem cells (ESCs) are pluripotent stem cells derived from early mammalian embryos.133 ESC chondrogenesis can be achieved by in vitro culture supplemented with growth factors.135 ESCs have been reported to improve cartilage repair in animal models.137 In 2009, the US FDA approved the world’s first clinical trial with human ESCs for the treatment of spinal cord injury,138 making it possible to translate ESCs for OA disease in the future.Regarding cell therapy, pluripotent stem cells have unlimited self-renewal and chondrogenic differentiation capacity,139 iPSCs have been successfully induced to differentiate into various cell types including chondrocytes.140–143 Notably, Wei et al. generated iPSCs from human OA chondrocytes and then induced them towards chondrogenic differentiation, suggesting the potential of OA chondrocytes for OA treatment.143Induced pluripotent stem cells (iPSCs) are another type of pluripotent stem cells generated directly from adult cells. iPSCs are more applicable than ESCs, as they can be derived from more donor tissues with less immunorejection, and have less ethical controversy.in vitro cell expansion and reimplant procedure, and is therefore regarded as a cost-effective and technically simpler alternative to current cell transplantation. The key factors to a successful cell homing process are the favorable cell niche that can be enhanced by excellent bioscaffolds, signaling biomolecules, and release technology.144 We have previously used a collagen type 1 scaffold containing stromal cell-derived factor-1 to create an in situ matrix environment.145 This microenvironment is conducive to the migration and adhesion of endogenous MSCs, thereby promoting the self-repair of partial thickness cartilage defects in a rabbit model. Another interesting study developed plasmid gene-activated osteochondral scaffold that could produce TGF-β1 for chondrogenic layer and BMP-2 for osteogenic layer.146 Endogenous BM-MSCs can be recruited and spatially controlled to simultaneously differentiate into chondro- and osteo-lineages within the scaffold. As OA usually affects complex tissues in the knee joint, this model may be exploited for future clinical treatment of OA disease.In terms of tissue-engineering strategies, more studies are now focusing on endogenous cell homing approaches. It aims at modifying a suitable microenvironment to recruit and mobilize the host cells from either the blood or a tissue-specific niche for self-repair. It avoids the costs, complexity, and risks involved in


Exosome nanoparticles carry a composite cargo, including microRNAs (miRs). Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG) surgery, we investigated if: 1) exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2) circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn), the current “gold standard” surrogate biomarker of myocardial damage.The concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed , non-cardiovascular (miR-122) and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs.The plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients. Observational data indicate that the abundance of released exosomes and their molecular cargo differ in response to environmental perturbations, including hypoxia[In vitro, exosomes have been shown to transfer biologically active components of their cargo into “recipient” cells, thus playing a role in cell-to-cell communication[in vivo difficult to explore. Nonetheless, it is possible to track nanoparticles in biological fluids,[Extracellular microRNAs (miRs) are resilient to degradation and thus are long-lived in biological fluids and measurable in both freshly collected and banked clinical samples. miRs, tg hypoxia, 8, chang hypoxia, and stig hypoxia. In vitrunication, 12. It unication, 14. In Interventional cardiologists have already provided evidence that cardiac expressed miRs  increase in the blood acutely following a myocardial infarction (MI) and some of these studies have additionally scrutinized the diagnostic potential of miRs by comparisons with cTns–17. We rbluebook.scts.org -Blue Book Online-Society for Cardiothoracic Surgery). CABG is intrinsically associated with variable degrees of myocardial damage related to the use of CPB and the ischemia-reperfusion insult of the cardioplegic arrest. We reasoned that this clinical setting would allow the determination of whether cardiac exosomes are released in response to myocardial injury and if this is sufficient to significantly affect the abundance and cargo of the pool of exosomes circulating in the peripheral blood. Moreover, the potential of circulating exosomes as novel biomarker of heart injury could be assessed by correlating circulating exosomes and elements of their molecular cargo with cardiac troponins (cTns), the “gold standard” surrogate biomarkers of myocardial damage[Coronary-artery-by-pass-graft (CABG) surgery using cardiopulmonary bypass (CPB) and myocardial cardioplegic arrest is one of the most commonly performed operations in the world  and the Imperial College Health Service Foundation Trust (ARCADIA). All patients provided written informed consent.Coagulation and Platelet Function Testing in Cardiac Surgery), a registered (ISRCTN20778544) UK National Institute of Health Research (NIHR)-funded single-centre prospective observational study, recruited 2,427 patients undergoing cardiac surgery at the Bristol Heart Institute. It has collected citrate plasma  before (prior to chest opening) and on completion of surgery (before chest closure). The former sample (pre-operation: pre-op) is taken before heparin administration and the latter (post-operation: post-op) within 30 minutes of reversal of heparin anticoagulation by protamine sulfate, when the patient is still in the operating theatre. The COPTIC research protocol was amended and ethically approved to perform microRNA (miR) and exosome analyses. In this study, we have used pre-op and post-op samples (banked at a temperature of between -70°C and -80°C) from a subgroups of male non-diabetic patients (n = 15), aged less than 75 years undergoing coronary artery bypass graft (CABG) surgery using cardiopulmonary bypass (CPB). See COPTIC (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) (REC 13/LO/1687) is a prospective observational study conducted at the Bristol Heart Institute and at the London Hammersmith Hospital. ARCADIA has been specifically designed to carry out analyses of non-coding RNAs (ncRNA), including miRs and extracellular vesicles (EVs), including exosomes, in patients undergoing first time CABG using CPB and cold blood cardioplegic arrest. Blood samples are taken at 4 time points: 1) in the anaesthetic room; 2) during surgery before establishment of CPB, and at 3) 24h and 4) 48h after the end of the operation. For this study, we used anonymized citrate plasma  and serum (for hs-cTn-I) samples from 6 male non-diabetic CABG patients previously stored at -80°C. See ARCADIA  machine (NanoSight). The plasma concentration of particles of a size (30 to 100nm) typical of exosomes was deteUsing a column-based system, exosomes were enriched from the ARCADIA patients’ plasma.Hs-cTn-I was measured in serial ARCADIA serum samples . The COPTIC samples were analysed with a non-hs cTn-I ELISA (Sigma) since the available hs-cTn-1 assay (and of other hs-cTn assays) is unsuitable for citrate plasma samples.post hoc Tukey’s test. Data are presented as the mean ± standard error of the mean (SEM). Comparisons between individual miR expression levels, exosome concentrations and loge hs-cTn-I concentrations were made using simple linear regression, with the regression coefficient (± SE) reported in each case to indicate the predictive power of the explanatory variable. Significance levels were determined using the F test, and data from all time points were analysed simultaneously. Statistical analyses were performed using STATA  and Prism . The level of significance was taken as p<0.05.For the COPTIC samples, regression analysis was used to investigate differences between pre- and post-cardiac surgery levels of extracellular miRs and exosomes. Results are illustrated graphically as scatter plots on a log scale with pre-surgery against post-surgery levels for either each miR or exosome concentration. Summary statistics are presented as mean difference, standard error (SE) and confidence interval for both pre- and post-surgery levels. For the ARCADIA samples, comparisons over each of the four time points were performed using repeated measures ANOVA with the The plasma nanoparticle concentration was analysed using a Nanoparticle Tracking Analysis (NTA) system . One μL of neat plasma was diluted with ultra-clean, sterile water to obtain a concentration of particles suitable to be read on the machine in a 1mL sample, according to the manufacturer’s guidelines. This was then passed through a flow cell at a constant flow rate using a syringe driver, where a laser beam was shone through the stream of particles. Once the temperature of the flow cell had stabilised at 25°C, 6 individual videos of 30 sec duration were recorded consecutively, with a 5 sec delay in between each. These videos were then processed using the NTA software, which uses the Stokes-Einstein Equation to determine the size of the particles from their Brownian motion. The plag for 30 min at 4°C to remove any remaining cell debris. The supernatant was transferred to a fresh tube and 5μL thrombin (500 U/mL) per 0.5 mL plasma was added to each sample, to remove the fibrin proteins. The samples were incubated at RT for 15 minutes while mixing, then centrifuged at 10,000 g for 5 min at RT. The supernatant was then passed through a sterile a 0.22μm filter  into a fresh tube. A sample volume of 100μL was applied to the top of the column and centrifuged at 500 g for 60 sec. After discarding the elute, 200μL of PBS was applied to the top of the column, the sample was centrifuge at 500 g for 60 seconds and the elute containing purified exosomes was stored at -20°C until required.Using a column-based system, Exo-spin Mini-Columns , exosomes were enriched from the ARCADIA patients’ plasma. Briefly, 100 μL of plasma prepared as described above was further centrifuged at 17,000 Exosomes from Patient 6 of the ARCADIA cohort were extracted using the ExoSpin columns as described above. Negative staining and TEM was carried out using an adapted, previously described protocol. Brieflyg for 15 min at 4°C and the supernatant fractions were used for Western blot. The protein content was measured using MicroBCA protein assay . Primary and secondary western blot antibodies are shown in Western blot analyses for exosome markers were performed on the plasma exosomal fractions extracted as above. Exosome preparations were lysed with RIPA buffer (Santa Cruz Biotechnology) with added protease inhibitor cocktail. Samples were centrifuged at 14,000 All the miRs measured in COPTIC and ARCADIA, together with their sequences, reasons for inclusion in the analyses (with literature references) and the miR assay codes are summarized in High Sensitive (hs) cardiac Troponin-I (cTn-I) (hs-cTn-I) was measured in serial ARCADIA serum samples by using the ARCHITECT STAT High Sensitive Troponin-I assay (Abbott). In the COPTIC samples, we measured cTn-I by ELISA (Sigma), because the ARCHITECT or other kits for either hs-cTn-I or hs-cTn-T do not work on plasma citrate samples.COPTIC (ISRCTN20778544) was a prospective observational study in patients undergoing heart surgery in which citrate plasma samples were collected at two time points: 1) in the anaesthetic room before surgery and 2) post-CPB immediately after heparin reversal before chest closure. In the COPTIC samples, plasma circulating nanoparticles of the size of exosomes increased immediately after completion of CABG, when the patients were still in the operating theatre . The anaWe next investigated the time-course of plasma miRs and exosomes response to cardiac surgery and changes in the exosomal miRs cargo. These tasks were developed using samples from a subgroup of CABG patients see .As shown in Next, we prepared an exosome-enriched fraction from aliquots of the same plasma samples employed for the analyses of above. We assessed the quality of our preparations. NTA showed that our preparation contained vesicles of the size (30 to 100 nm) of exosomes . AdditioInterestingly, the pattern of miR changes associated with CABG was different when miRs were measured in the plasma exosomal fraction rather than in whole plasma . In deta-14 ± 2.0x10-14, P<0.0001) (CTn-I was unchanged at completion of surgery (data not shown). However, the hs-cTn-I concentration increased at 24h and 48h post-CPB . The who<0.0001) . The con<0.0001) . Finally<0.0001) .This study provides novel evidence that: 1) the concentration of cardiac-enriched, ischemia-responsive miRs and of nanoparticles of exosome size increases in the plasma early after CABG; 2) miRs of possible cardiac origin circulate as part of exosomes and in non-exosomal plasma fractions, with the exosome to whole plasma concentration ratios of individual miRs differently affected by surgery; 3) the concentrations of exosomes and of exosomal cardiac miRs after CABG are positively correlated with the concentration of hs-cTn-I; 4). Following CABG, exosomes increased in the peripheral circulation earlier than cardiac troponins.Based on the above data, we propose that the heart-derived exosomes that circulate in the peripheral blood may be reporters of the myocardial injury, thus enabling for diagnosis and monitoring of cardiac patients. Consequently, we propose that blood exosome-based analyses should be further considered in the attempt to develop novel semi-invasive biomarkers of cardiac damage. Analyses of blood exosomes and exosomal miRs hold several properties that make them particularly interesting. From a mechanistic standpoint, exosomes are actively secreted from living cells, 4–6 andper se, be a biomarker of the response of the patients to the intervention, possibly enabling prediction of clinical outcomes. This possibility requires validation in larger cohorts of patients. In case of a positive result, the measurement by NTA of the exosome-sized particle concentration in the blood could suffice. Of note, this method is relatively cheap and fast and requires an extremely small volume of biological fluid[In this study, we have shown that the plasma concentration of exosome-sized nanoparticles is highly positively correlated with hs-cTn and that increases in plasma exosomes precede the appearance of detectable cTn in the blood after CABG. Hence, in the cardiac surgery and interventional cardiology settings, changes in the blood exosome concentration after a procedure could, cal fluid. HoweverExosomes are released by a variety of cell types and hence changes in their plasma concentration cannot be assumed to be dependent solely on their release from the heart cells. However, in the setting of cardiac surgery, the cardiomyocyte contribution to the circulating exosomes is strongly suggested by the presence of myocyte-enriched miRs  in the plasma exosome cargo and in the fact that abundance of exosomal cardiac miRs increased after CABG.Flow cytometry analyses have already been developed to understand the cellular origin of microparticles, which are bigger than exosomes. EquivalWe did not observe any association between miR levels in whole plasma and hs-cTn-I in our CABG patients. Previous reports in the literature are inconsistent with respect to associations between individual cardiac-enriched miR concentrations in the whole plasma and cTns, 45–47. via exosomes, while miR-1 and miR-133 are released via exosomes and exosome-independent mechanisms in similar proportions. The reasons beyond these differences cannot be inferred from the current data and require further investigation aiming to clarify the mechanisms governing exosome biogenesis (including exosome miR cargo formation) and secretion from the heart under homeostatic conditions and after a “cardiac stress”, such as ischemia/reperfusion.We have found that after surgery, miR-24 and miR-210 were substantially enriched in the total pool of plasma exosomes. In contrast, the exosome/whole plasma concentration ratios of miR-1, miR-133a and miR-133b were mostly unchanged. This suggests the possibility that after CABG miR-24 and miR-210 are predominantly released In conclusion, the plasma concentrations of exosomes and their cargo of cardiac miRs change significantly in patients undergoing CABG. The circulating exosome numbers and their cargo of cardiac miRs are associated with hs-cTnI release. Moreover, the change in nanoparticle concentration precedes the cTn-I increase. Our findings suggest the trafficking of exosome-mediated miRs from the heart, which is amplified by cardiac surgery. Moreover, the data suggest a potential role for exosomes as new biomarkers of myocardial injury.S1 Data(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Further analysis found that miR-572 could regulate the expression of NCAM1 in the hippocampal neurons and interfering miR-572 expression could facilitate the restoration of cognitive function in vivo. Moreover, clinical correlation analysis found that the miR-572 expression was associated with the incidence of POCD. Collectively, miR-572 is involved in the development and restoration of POCD and it may serve as a biological marker for early diagnosis of POCD.Post-operative cognitive dysfunction (POCD) is a commonly-seen postoperative complication in elderly patients. However, the underlying mechanisms of POCD remain unclear. miRNAs, which are reported to be involved in the pathogenesis of the nervous system diseases, may also affect POCD. In this study, miRNA microarray technology was used to analyze the circulating miRNA expression profile of POCD patients. Among the altered miRNAs, miR-572 had the greatest decrease, which was also verified  Human cognition is an important part of neuroscience with intriguing findings and immense implications recently . With dePost-operative cognitive dysfunction (POCD) is a commonly-seen postoperative complication in elderly patients , 4. POCDN-methyl-D-aspartate (NMDA) receptor- associated neurobehavioral disorders [MicroRNAs (miRNAs) are widely distributed in eukaryotes and regulate the translation and transcription of target genes by degrading target mRNAs or inhibiting the translation of target genes . Recentlisorders , and abnisorders .Recent studies have found that the miRNAs in the circulating peripheral blood are similar to the miRNAs in tissues , 16. As Blood samples were obtained from 38 POCD patients including 29 cases with restoration of cognitive function 3 month after surgery and 9 cases without  in the DInclusion criteria were age >60, surgery for knee or hip replacement under spinal anesthesia, American Society of Anesthesiologists physical status (ASA) 1–3, and patient consent. Exclude criteria were >85, ASA physical status >3, neurological disorders, stroke or other affective central nervous system disease, history of serious psychiatric illness, long-term use of psychoactive substances, multiple trauma and the presence of head injury. Demograth and 30th postoperative day, using the MMSE test combined with the Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS) and the American Psychiatric Association Diagnostic and Statistical Manual (DSM-IV). Reduction of MMSE score by ≥ 2 points on the 7th postoperative day compared with preoperative day was considered evidence of cognitive decline [Cognitive function was assessed on the day before the surgery and on the 7 decline , 20. The decline .All samples collection was performed with the patients' consent, and signed informed consent was obtained. The study was approved by the Ethics Committee of Changhai Hospital Second Military Medical University. The peripheral blood samples (5 ml) were collected into the anticoagulant tubes at 1 day before surgery, 24 h after surgery, and 7 days after surgery and were centrifuged ; the supernatant was stored at—70°C until testing.HT-22 cells, a widely used hippocampal neuronal cell line derived from parent HT4 cells, were maintained in Dulbecco's modified Eagle's medium (DMEM) supplied with 10% FBS, as previously described [miRNA microarray analyses were performed by Shanghai Biotech Corp. using blood from POCD patients. Briefly, 3 patients . Reverse transcription reactions were conducted with oligo (dT)18 primers and random primers according to the instructions of the manufacturer of the M-MLV Reverse Transcriptase kit (Invitrogen). MiRNAs reverse transcription was performed using miRcute miRNA First-strand cDNA Synthesis kits (TIANGEN) according to the manufacturer’s instructions. Real-time PCR was performed with SYBR Premix Ex TaqTM (TaKaRa) using the StepOnePlus Real-Time PCR system . The primers used in these assays are listed in Total soluble proteins (100 μg) extracted from the HT-22 cells were resolved in 10% SDS-polyacrylamide gels and transferred electrophoretically to a polyvinylidene fluoride membrane. Blots were blocked with 5% skim milk, followed by incubation with NCAM rabbit polyclonal antibody  or β-actin . Blots were then incubated with goat anti-mouse or anti-rabbit secondary antibody (Santa Cruz Biotech. Inc.) and visualized by enhanced chemiluminescence.The aged male rats  were purchased from the Transgenic Animal Research Center of the Second Military Medical University. All mice were maintained in a pathogen-free facility and used in accordance with the the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The study was approved by the Ethics Committee of Changhai Hospital Second Military Medical University. According to the method described previously, the aged rats  were anesthetized by inhalating 1.5% isoflurane and underwent a splenectomy to establish the POCD model which was used widely to mimic POCD in elderly human patients –25. The The rats were trained for the water maze at 1 day before surgery, and the water maze test was performed 24 h and 7 days after the surgery; and the subsequent data were used as the learning and memory score , 27.Probes of pri-miR-572 with a digoxin label were synthesized for in situ hybridization (ISH). ISH was performed on sections of tissues as previously reported. In situ hybridization is a necessary experimental complement to microRNA (miRNA) expression profiling in the human brain.P<0.05 was considered statistically significant.Student’s t-tests were used to compare two groups unless otherwise indicated (χ2 test). Categorical data were analyzed using Fisher’s exact test, and quantitative variables were analyzed using t-tests or Pearson’s correlation test. To screen microRNA associated with cognitive function restoration in patients with POCD, the circulate miRNA expression profiles were analyzed using microRNA microarray as mentioned in the Method. The microarray results showed that, compared with the preoperative microRNA expression profile, there were a large number of abnormally down-regulated circulating miRNA molecules in the peripheral blood 24 h after surgery . Clusterleft), and named it rno-miR-572. Moreover, we used in situ hybridization to detect rno-miR-572 expression in the rat hippocampal tissues (right). Using the method reported by Barrientos et al. we performed a splenectomy in aging rats. The water maze test illustrated that, compared with before surgery, the latency of boarding the platform of animals receiving a splenectomy was significantly prolonged (left). In addition, rats without cognitive dysfunction showed no significant changes of miR-572 expression (right). The results were consistent with those of clinical specimens, suggesting that miR-572 might be involved in regulating cognitive function changes in the POCD rat model. Because miRNAs mainly play a negative regulatory role, we hypothesized that in the early stage of POCD, the body might down-regulate miR-572 to up-regulate its downstream target genes, thus contributing to the restoration of cognitive function. Therefore, we selected rats with reduced cognitive function and used a lateral ventricular micropump to inject the miR-572 inhibitor to further validate the above hypothesis. After 3 days, the water maze test found that, compared with the control (pLKO-anti-mock), rats injected with pLKO-anti-572 showed significantly shorter latency to board the platform, suggesting significantly improved cognitive function . The results suggested that hsa-miR-572 has 9 potential downstream target genes . Among tTo test whether miR-572 can be used as a marker for the early diagnosis and prognosis of POCD, we collected the peripheral blood from elderly POCD patients (n = 38) before and 24 h and 7 days after surgery. Real-time quantitative PCR was used to measure the miR-572 expression in the peripheral blood samples. The results showed that the blood miR-572 levels in POCD patients (n = 38) at 24 h (1.21 ± 0.79) and 7 days (0.724 ± 0.883) after the surgery were significantly lower than the preoperative level (1.42 ± 1.06) . The perP>0.05) in the neurocognitive function tests at 3 months after the surgery. Nine patients (Group 2) failed to restore the preoperative level and had varying degrees of residual cognitive impairments . Quantitative PCR showed that the blood miR-572 expression levels in these 9 patients were significantly higher at 24 h and 7 days after the surgery than those of the other POCD patients  was used to assess the cognitive function of patients with POCD. We found that the performances of most patients  restored to the preoperative level Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis. Sepsis is a systemic inflammatory response syndrome caused by the inability of the host to locally restrict an infection . Sepsis The successful clearance of pathogens to avoid their spreading into the circulation depends on efficient neutrophil recruitment to the infection site and microbicidal activity of the emigrated cells. Neutrophil migration to the infection site is mainly mediated by G protein-coupled receptor signaling, including the CXCR subfamily, the leukotriene receptor and the C5a receptor , 4. MoreA frequent complication of sepsis is the development of organ dysfunction, including that of the heart, kidney and liver . The patDuring their activation, neutrophils also release a network of chromatin fibers associated with granule antimicrobial peptides and enzymes, such as MPO, elastase, and cathepsin G, known as neutrophil extracellular traps (NETs) . NETs reIn the present study, we showed that NETs were systemically formed in sepsis and play a dual opposite role: on the one hand, NETs control the infection, but on the other hand, they induce tissue and organ damage. Accordingly, the degradation of NETs by recombinant DNase (rhDNase) increased the bacterial burden, but when combined with antibiotics to control the infection, this strategy attenuated the systemic inflammatory response, reduced endothelial and organ damage, and increased the survival rate of severely septic mice. Additionally, we present the first report that serum concentrations of cf-DNA, a surrogate marker of NETs levels, were positively correlated with sepsis severity and organ failure in human sepsis.Peripheral blood samples were collected from 31 septic patients who were prospectively enrolled in the study within the first twenty-four hours of admission in the Emergency Department of a high-complexity hospital. All patients presented clinical or laboratory variables that fulfilled the criteria for sepsis . WrittenC57BL/6 mice were obtained from the Ribeirão Preto Medical School. All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was previously approved by the Animal Ethics Committee of the Ribeirão Preto Medical School, University of Sao Paulo, Sao Paulo, Brazil (Protocol number: 047/2010).The following drugs were used: ertapenem sodium ; recombinant human DNase ; and Escherichia coli lipopolysaccharide . Ertapenem sodium and LPS were dissolved in phosphate-buffered saline (PBS).Sepsis was induced by cecal ligation and puncture (CLP) as described elsewhere . BrieflyFor all survival experiments, the survival rate was determined every 12 h for 7 days. At this time, mice that showed signs of imminent death  were euthanized by ketamine/xylazine  overdose. At the end of 7 days, live mice were euthanized. The number of animals for survival curves was 5–10 per each group. For sample harvesting, mice were euthanized in a CO2 chamber.Protocol 1—For the characterization of organ damage during sepsis, we used two experimental groups: S\sham and CLP-SS (mice with severe sepsis induced by CLP). Blood levels of the organ damage biomarkers creatine kinase MB isoenzyme (CK-MB), blood urea nitrogen (BUN) and aspartate aminotransferase (AST), tumor necrosis factor (TNF), cell-free DNA (cf-DNA), neutrophil extracellular traps (cf-DNA/MPO) and bacteria were determined at 3, 6, 12, or 24 h after sepsis induction. Neutrophil infiltration in lung tissue was estimated by MPO quantification at 3, 6 or 12 h after sepsis induction. There were 5 animals in each group and the experiment was repeated 2 times.Protocol 2 –For the experiment with rhDNase treatment, we used three experimental groups: sham, CLP + Sal  and CLP + rhDNase (severe sepsis treated with rhDNase sc.) The mice were treated with saline or rhDNase  pre- (10 min) and post- (4 h) sepsis induction. Six hours after sepsis induction, we evaluated the blood levels of organ damage biomarkers , cf-DNA/NET, and bacteria. In survival experiments, mice were treated as previously described and every eight hours after that. There were five animals per group and the experiment was repeated 2 times.Protocol 3 –For the experiment with rhDNase treatment plus antibiotic therapy, we used three experimental groups: sham, CLP + Ant  and CLP + Ant + rhDNase (severe sepsis treated with antibiotic plus rhDNase). Mice were treated with saline or rhDNase  plus ertapenem antibiotic . Twelve hours after sepsis induction, we evaluated the blood levels of organ damage biomarkers , cf-DNA/NET, TNF, IL-6, and MPO in lung tissue, and bacteria levels in the blood. We also quantified the bacterial load and the numbers of mononuclear leukocytes and neutrophils in the peritoneal lavage, as described elsewhere . For theProtocol 4 –For the experiment with endotoxemic mice treated with rhDNase, we used three experimental groups: CTRL (naive), LPS + Sal  and LPS + rhDNase (mice with endotoxic shock treated with rhDNase sc.). The mice were treated with saline or rhDNase  pre- (10 min) and post- (8 h) endotoxic shock. Twelve hours after endotoxic shock induction, we evaluated the blood levels of organ damage biomarkers , cf-DNA, TNF-α and MPO in lung tissue. We also evaluated the NETs deposition in kidney tissues 12 h after endotoxic shock induction. For the survival experiments, the mice were treated with saline or rhDNase  pre- (10 min) and post- (8 h) endotoxic shock and then every 8 h thereafter up to day 3. There were 5 animals in each group and the experiment was repeated 2 times.in vivo, which is equivalent to 100 μg/mL. Cell free-DNA was quantified in the serum using the Quant-iT™ PicoGreen® kit (Invitrogen) according to the manufacturer’s instructions, as previously described [Protocol 5 –For the experiment to determine whether rhDNase degrades cf-DNA in vitro in the serum of septic patients, 40 μL of serum was incubated with rhDNase (concentration: 100 μg of DNase/1 mL of serum) for 30 minutes at 37°C. This concentration was determined based on the corresponding concentration of DNase that was experimentally tested escribed . There wThe bacterial counts were determined as previously described . BrieflyCytokine and chemokine concentrations were measured by ELISA, as previously described . The detThe animals were anesthetized and whole blood was collected by cardiac puncture. The samples were centrifuged , and the serum or plasma was collected. Cell free-DNA (cf-DNA) was quantified in the serum using the Quant-iT™ PicoGreen® kit (Invitrogen) according to the manufacturer’s instructions, as previously described . The fluThe concentration of cf-DNA bound to MPO (cf-DNA/MPO), a constituent of NET, was also measured in the serum or plasma of septic mice. This method allowed the identification of NETs in the circulation of the animals. Briefly, an antibody bound to the 96-well flat-bottom plate captured the enzyme MPO, a constituent of NET, and the amount of DNA bound to the enzyme was quantified using the Quant-iT™ PicoGreen® kit (Invitrogen), as above.The animals were anesthetized and whole blood was collected by cardiac puncture. The samples were centrifuged  and the serum was collected. The following biochemical parameters were measured in the serum as markers of multiple organ injury or dysfunction: liver injury was assessed by measuring the increase in serum concentrations of AST; renal dysfunction was evaluated by measuring the increase in BUN; and cardiac lesions were assessed by measuring CK-MB levels. The determinations were made using a commercial kit . Serum endocan concentrations were measured by ELISA . The detection limit of the test for endocan was 1.46 pg/mL.Briefly, the animals were euthanized in a CO2 chamber after sepsis or endotoxemia induction. The myeloperoxidase activity in lung homogenates was assayed as previously described .Samples of kidney tissue were collected from mice, embedded in Tissue-Tek® O.C.T.™, immediately frozen in 2-methylbutane (isopentane) resting in liquid nitrogen and stored at −70°C. Sections were cut to a thickness of 10–15 μm, fixed with 4% paraformaldehyde and stained with Hoechst 33342 , anti-MPO  and anti-DNA/histone H1  antibodies, followed by anti-rabbit Alexa Fluor 594  or anti-mouse Alexa Fluor 488 . Confocal images were taken with a Leica TCS SP5-AOBS microscope ; epifluorescence images were taken with a Zeiss Axioplan. Permeabilizing agents were not used, resulting in exclusive extracellular labeling. Assessment was made in a purely qualitative fashion to detect the presence or absence of NETs in the analyzed samples.The animals were euthanized 12 h after endotoxic shock induction, and the lungs, hearts, and livers were isolated and fixed by immersion in 10% paraformaldehyde, dehydrated and embedded in paraffin wax. Then, 5-μm-thick sections were stained with hematoxylin and eosin for histological examination. A semi-quantitative analysis was carried out in a blinded fashion by an experienced pathologist who was unaware of the groups  to quantify the results, defined as: 0 = normal, + = mild, ++ = moderate, +++ = severe histological changes. The tissues and parameters assessed included the heart ; the lungs ; and the liver .All of the data are reported as the means ± SEM of the values obtained from two independent experiments . The data were compared by analysis of variance (ANOVA) followed by the Bonferroni correction. Tukey’s test or Student’s t-test were used for unpaired values. Bacterial counts were analyzed by the Mann-Whitney U test. The survival rate was expressed as the percentage of live animals, and the Mantel-Cox log-rank test was used to compare the survival curves. Clinical data were summarized using the means ± SD or medians with interquartile ranges (IQRs). If variables were skewed, nonparametric tests were employed. Continuous variables were compared using t-tests, nonparametric tests for trends across ordered groups, the Mann-Whitney U-test, and the Kruskal-Wallis test. Spearman’s rank correlation coefficient (σ) was calculated to describe correlations between cf-DNA/NET plasma concentrations and clinical variables. Differences were assumed to be statistically significant at a level of 5%.We investigated the systemic formation of NETs in mice following CLP-induced sepsis. As shown in To verify whether NETs were involved in the development of organ dysfunction and control of bacterial growth in our experimental conditions, we investigated whether the degradation of DNA, the major constituent of NETs , affecteNext, we asked whether post-treatment of septic mice with rhDNase combined with antibiotics to control the infection would allow us to determine a possible role for NETs in sepsis-induced organ injury. Thus, mice subjected to severe sepsis were treated with ertapenem  alone or with ertapenem plus rhDNase . We observed that the septic mice treated with antibiotics had reduced blood bacterial counts compared to septic mice treated with saline . CombineTo confirm our results, we investigated whether the rhDNase treatment could decrease organ dysfunction in LPS-induced endotoxic shock, a model without live bacteria . For thaWe next performed histological analyses to confirm the biochemical markers of lesion findings. Normal (score 0) heart , lungs  and liveFurther, we evaluated whether NETs are present in sepsis-related injured tissues. For that, we performed immunofluorescence and confocal microscopy analysis of kidney tissues 12 h after the induction of endotoxic shock. We found the typical components of NETs, i.e., DNA/histones  and MPO To assess whether our experimental observations could be extended to the clinical setting, we quantified the serum concentrations of cf-DNA from healthy control volunteers and septic patients. We prospectively included 31 patients within the first twenty-four hours of admission in the Emergency Department of a high-complexity hospital. Eight healthy volunteers were included as controls. The baseline demographic and clinical characteristics are summarized in Consistent with the results obtained in murine experiments, septic patients showed higher serum concentrations of cf-DNA than healthy control volunteers . We did In this study, we demonstrated that cf-DNA/NETs are present in septic patients and in mice with CLP-induced experimental sepsis. We noticed that when cf-DNA, a major constituent of NETs structure, was degraded in CLP-induced sepsis by rhDNase treatment, the animals began to die earlier than in the control group, possibly due to a blockage of bacterial burden control in those animals. This observation is in agreement with a previous study reporting that NETs depletion precludes the early immune response and aggravates polymicrobial sepsis . MoreoveTo circumvent this issue, we decided to combine rhDNase treatment with a broad-spectrum antibiotic (ertapenem) to avoid a failure of bacterial control. This strategy prevented the rhDNase-related increase in the bacterial burden, decreased sepsis-induced organ damage, and increased the overall survival rate of septic mice. All assessed parameters of organ injury , endothelial damage (endocan), systemic inflammation (serum TNF-α and MPO in the lungs) and histopathological changes were improved by the combined treatment, demonstrating that the effects are not organ specific. Additionally, we evaluated the inflammatory response at the focus of infection and did not observe any difference in the number of mononuclear cells or neutrophils. These results indicate that the improved outcome of septic mice treated with antibiotics plus rhDNase was not due to better control of the local infection but instead due to a systemic effect of the treatment. Accordingly, the decrease in NETs following rhDNase treatment also reduced the levels and intensity of inflammatory and organ injury parameters in the aseptic model of LPS-induced endotoxic shock. Similarly, other studies also demonstrated that rhDNase treatment reduced organ injury in CLP- or LPS-induced sepsis , 41. HowTo measure cf-DNA, a major constituent of NETs and cf-DNA associated with MPO (cf-DNA/MPO), another constituent of NETs, we used two methodologies and demonstrated that, in addition to the levels of free DNA (cf-DNA) being higher in relation to the levels of cf-DNA/MPO in the serum, both methods show similar results for the release after sepsis induction. Thus, our data indicate that the use of the cf-DNA method can be a surrogate to indicate NETs formation. In contrast to our results, Hamaguchi and colleagues have previously demonstrated that NETs may not be the primary source of cf-DNA and hypothesize that necrotic tissues are responsible for cf-DNA release . Howeverin vitro, promoting damage to themselves [NETs are highly related to endothelial damage and organ failure, which are important events in sepsis. Activated endothelial cells induce the formation of NETs emselves , 43. Addemselves . Notwithemselves . Althougemselves , 45, 46.emselves . Our resemselves . Howeverin vivo NETs formation in septic patients. In this context, to the best of our knowledge, we are the first to describe that serum NETs are positively correlated to clinical parameters of sepsis severity and organ injury. We demonstrated that septic patients who presented moderate or severe acute kidney injury  have increased circulating serum NETs compared to those without acute kidney injury or with only mild disease (AKIN grade 1). Additionally, NETs levels were higher in subjects that developed severe or moderate ARDS compared to those with mild ARDS or without ARDS. Accordingly, NETs were previously related to ARDS development in patients with influenza pneumonitis [Hashiba and collaborators demonstrated that NETs generation by neutrophils from septic patients is downregulated compared to neutrophils from nonseptic patients , which cumonitis .Some potential limitations of this work include the use of an experimental model with 100% mortality, which does not perfectly model the general clinical setting. However, it is important to note that even in this severe experimental condition, the blockage of NETs presented a marked protective effect. One can also argue that another limitation was the use of cf-DNA as a surrogate to NETs in a number of different assays, although we have demonstrated that they strongly correlate with each other. Because we included septic patients from a high-complexity tertiary and reference hospital, a selection bias might have occurred, which resulted in an observed high mortality rate. The control group was healthy volunteers instead of non-septic sick patients. For this reason, based on our data, NETs cannot be used as a biomarker to discriminate septic from non-septic patients. However, as mentioned previously, our data support the hypothesis that NETs levels separate patients with more severe organ-specific damage from those with less severe disease. Moreover, the study power was not strong enough to test the hypothesis that NETs is correlated with mortality in septic patients.We showed that NETs are present in sepsis and participate in sepsis-induced organ failure. These results encourage us to support the evaluation of serum (or plasma) NETs concentration as a predictive biomarker for the early assessment of sepsis severity. Moreover, our experimental findings lead us to envision a phase I/II clinical trial to test whether degrading cf-DNA/NET through rhDNase administration could improve sepsis outcomes in patients.Our data indicate that cf-DNA/NETs are involved in the severity of sepsis in mice and humans. Although NETs play a beneficial role in the killing of pathogens, their involvement in sepsis-induced organ injury is deleterious. Therefore, the strategy of rhDNase degradation of cf-DNA coupled with antibiotic therapy might represent a novel therapy for the management of sepsis and the prevention of organ dysfunction.S1 Figsc.). Plasma concentrations of cf-DNA 6 and 12 h after sepsis induction and rhDNase treatment (last two bars). (B) Blood samples were collected 6 hours after sepsis induction and cf-DNA levels were determined in plasma and serum. * p < 0.05 compared with the sham group . (C) Blood samples were collected 6 hours after sepsis induction, and NETs were determined in plasma and serum. * p < 0.05 compared with the sham group .Mice were subjected to sham or CLP-induced severe sepsis. At the indicated time-points, cf-DNA (A-B) or NET (C) were measured in plasma or serum, as indicated in the figure. (A) Blood samples were collected 3, 6, 12 and 24 hours after sepsis induction, and plasma concentrations of cf-DNA were determined . * p < 0.05 compared with the sham group . Animals were treated pre-sepsis (10 min) and post-sepsis (4 h) with Sal (control) or rhDNase Click here for additional data file.S2 FigMice were subjected to sham surgery or CLP-induced severe sepsis. At the indicated time-points, blood bacterial levels (A), serum TNF (B), MPO in lung tissue (C) and serum concentrations of CK-MB (D), BUN (E) and AST (F) were determined. The data are reported as the mean ± SEM. *p < 0.05 compared with the sham group; # p <0.05 compared with the 3 h group . Bacteria: * p < 0.05 compared the sham group; # p < 0.05 compared with the 3 h group . (G) Survival rates of mice subjected to CLP. * p < 0.05 compared with the sham group .(TIFF)Click here for additional data file.S3 FigMice were subjected to sham surgery or CLP-induced severe sepsis. The mice were post-treated with saline or rhDNase  with ertapenem antibiotic . Twelve hours following sepsis induction, the numbers of mononuclear cells (A) and neutrophils (B) were determined in the peritoneal lavage. * p <0.05 compared with the sham group .(TIFF)Click here for additional data file.S1 Table§ NETs (neutrophil extracellular traps) serum concentrations are expressed as μg/mL. Bilirubin and creatinine serum concentrations are expressed as mg/mL.(DOCX)Click here for additional data file.


The present study aimed to evaluate regional liver function impairment following transcatheter arterial chemoembolization (TACE), assessed by magnetic resonance imaging (MRI) enhanced by gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). Additionally, this study evaluated the associations between signal intensity and various clinical factors. A prospective study was conducted between March 2012 and May 2013 with a total of 35 patients. Gd-EOB-DTPA-enhanced MRI was performed 3–5 days after TACE therapy. The signal to noise ratio (SNR) was subsequently calculated for healthy liver tissue regions and peritumoral regions, prior to and 20 min after Gd-EOB-DTPA administration. The correlation between clinical factors and relative SNR was assessed using Pearson’s correlation coefficient or Spearman’s rank correlation coefficient. Prior to Gd-EOB-DTPA administration, the SNR values showed no significant difference  in healthy liver tissue regions  compared with peritumoral regions . On measuring at 20 min following Gd-EOB-DTPA administration, the SNR in healthy liver tissue regions  was significantly higher  compared with that in peritumoral regions . The relative SNR in peritumoral regions correlated only with the quantity of iodized oil used during TACE therapy ; the age, gender, diameter and blood supply of the tumor, or Child-Pugh class of the patient did not correlate with relative SNR. Gd-EOB-DTPA-enhanced MRI may be an effective way to evaluate regional liver function impairment following TACE therapy. The overeatment –13. The Written, informed consent was obtained from all patients. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of The Second XiangYa Hospital of Central South University .A total of 35 HCC patients who underwent Gd-EOB-DTPA-enhanced MRI of the liver were enrolled in this prospective study between March 2012 and May 2013. Of the 35 patients, four were excluded from the study due to poor postoperative clinical status and two were excluded due to poor breath-holding during MRI examination. The final study population comprised 29 patients .The diagnosis of HCC was determined on the basis of the following criteria: Typical lesions observed on at least one imaging modality  with an elevated serum α-fetoprotein level  or liver biopsy with pathological confirmation of HCC (n=12).2).Inclusion criteria were as follows: Patients who could understand the study documents , had a single lesion with tumors <10 cm in size; were categorized as Child-Pugh class A or B prior to surgery and were receiving TACE therapy for the first time. Exclusion criteria were as follows: Severe motion artifacts due to poor breath-holding, profound liver cirrhosis, multiple lesions, tumors >10 cm in size, obstruction of main or first branch of portal vein, difficulty in locating the feeding artery during the procedure or hepatic artery to portal vein shunting. Exclusion criteria for the use of Gd-EOB-DTPA were as follows: Allergy to Primovist, severe cardiovascular disease or kidney insufficiency  bidirectional digital subtraction angiography. Using the Seldinger technique , a catheMRI was performed 3–5 days after TACE therapy using a 3T superconducting MRI system  with a phased array body coil  and the following imaging parameters: 7 mm section thickness and 3 mm intersection gap. Three-dimensional T1-weighted turbo field echo sequence with spectral presaturation inversion recovery fat suppression  was utilized pre-contrast and post-contrast . Respiratory-triggered T2-weighted fast spin echo sequence with short TI inversion recovery fat suppression  was used prior to injection of the contrast agent. The contrast agent was used at a dose of 0.025 mmol/kg body weight and at an injection rate of 2 ml/s by 20 ml saline flush using an intravenous line .In the evaluation of hepatocytic uptake of Gd-EOB-DTPA, signal intensity (SI) of the region of interest (ROI) of the liver was measured by one radiologist with 20 years experience of abdominal imaging, who was blinded to the clinical data and selection of ROIs. The ROIs were selected to be as large as possible, avoiding large vessels and biliary ducts, and the identical location was used prior to and following Gd-EOB-DTPA administration. Each ROI was circular or oval. Signal to noise ratio (SNR) was calculated for peritumoral and healthy liver tissue regions , by diviafter - SNRbefore)/SNRbefore] × 100.The relative SNR in the peritumoral regions was measured to assess the correlation between hepatocytic uptake of Gd-EOB-DTPA and potential clinical influencing factors . Relative SNR = [ and were analyzed using SPSS 19.0 software . The correlation between clinical factors and relative SNR was analyzed using Pearson’s correlation coefficient or Spearman’s rank correlation coefficient. Pearson’s correlation analysis was used to evaluate the association between the relative SNR of liver parenchyma and the age of the patient, the diameter of the tumor and the quantity of iodized oil used for TACE therapy. Spearman’s rank correlation was used to evaluate the association between the relative SNR of liver parenchyma and clinical parameters including gender, blood supply of the tumor and Child-Pugh class. The quantitative parameter of healthy liver tissue regions vs. peritumoral regions was calculated using a paired Of the 29 patients, 22 were categorized as Child-Pugh class A and seven as Child-Pugh class B. The quantity of iodized oil used for individual patients varied from 5–30 ml, with a mean quantity of 16.14±7.04 ml. A poor blood supply to the tumor was observed in nine patients, while a rich blood supply was observed in the remaining 20. The diameter of the tumors ranged from 3.1–9.9 cm, with a mean diameter of 6.94±2.15 cm. No correlation was observed between the blood supply and the diameter of the tumor . Detailed clinical information is listed in t=1.341, P=0.191). When measured 20 min after the administration of Gd-EOB-DTPA, the SNR values of healthy liver tissue regions  were significantly higher compared with those of the peritumoral regions . In peritumoral regions, the SNR also exhibited a significant increase when measured 20 min after Gd-EOB-DTPA administration  compared with that prior to administration. The absolute change in SNR for healthy liver tissue regions was significantly higher  compared with those of the peritumoral regions  compared with those of peritumoral regions, gender  or Child-Pugh class  of the patient. Additionally, no correlation was observed between the SNR and the blood supply  or the diameter  of the tumor. Relative SNR, measured 20 min following Gd-EOB-DTPA administration, was observed to correlate only with the quantity of iodized oil used during TACE therapy  for their activity . These tet al , which limited the power of the data analysis. Secondly, histological confirmation of regional hepatic damage was not included. The impairment of the liver tissue may have been caused by procedure-related cell death, parenchymal hypoperfusion and infarction of the liver tissue; histological examination is required to determine the actual cause of regional hepatic damage at a cellular level. Finally, as the patients may have had different underlying diseases, a bias was introduced by the potential varying disease mechanisms. In future studies, homogenous patient populations should be utilized.In conclusion, the present data demonstrates an apparent decrease in Gd-EOB-DTPA uptake in peritumoral liver regions compared with healthy liver tissue regions 20 min after Gd-EOB-DTPA administration, indicating the existence of regional hepatocytic injury caused by TACE therapy. Gd-EOB-DTPA-enhanced MRI may therefore represent an effective, non-invasive tool for evaluating regional liver function impairment following TACE therapy. Furthermore, an association was observed between the relative SNR and the quantity of iodized oil used, indicating that the dosage of oil used may impact the subsequent level of functional liver impairment. Further study should be concerned with the timing of liver function recovery after TACE therapy using Gd-EOB-DTPA-enhanced MRI.