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Fibrosis is the main cause of limited range of motion (ROM) of shoulder in patients with frozen shoulder (FS). Overexpression of Interleukin 6 (IL-6) has been correlated with pathogenesis of FS. However, the underlying mechanism remains largely unexplored. In the current study, we focused on isolating synovial fibroblasts of FS and determining the influence of IL-6 as well as PI3K-Akt signaling pathway on the fibrotic process of synovial fibroblasts in FS by using RNA Sequencing (RNA-seq) and other molecular biology techniques. Synovial fibroblasts of FS express more extra cellular matrix (ECM) than that of control. RNA-seq results and bioinformatic analysis indicate that PI3K-Akt signaling pathway play an important role in the fibrotic process of FS, and IL-6 is the most related gene among those related to this process. The expression levels of IL-6 / IL-6R in FS synovial fibroblasts and IL-6 in culture supernatant were both significantly increased. siRNA interference with the expression of IL-6 attenuates the fibrosis level of FS as well as phosphorylation level of Akt. The findings suggest that synovial fibroblasts are key effector cells of fibrosis of FS. Activation of PI3K-Akt pathway can promote fibrosis of synovial fibroblasts in FS. IL-6 is up-regulated in synovial fibroblasts of FS and promoted the FS fibrosis through PI3K-Akt signaling pathway.

Mesenchymal stem cells (MSCs) are a common kind of multipotent cell in vivo, but their heterogeneity limits their further applications. To identify MSC subpopulations and clarify their relationships, we performed cell mapping of bone-marrow-derived MSCs through single-cell RNA (scRNA) sequencing. In our study, three main subpopulations, namely, the stemness subpopulation, functional subpopulation, and proliferative subpopulation, were identified using marker genes and further bioinformatic analyses. Developmental trajectory analysis showed that the stemness subpopulation was the root and then became either the functional subpopulation or the proliferative subpopulation. The functional subpopulation showed stronger immunoregulatory and osteogenic differentiation abilities but lower proliferation and adipogenic differentiation. MSCs at different passages or isolated from different donors exhibited distinct cell mapping profiles, which accounted for their corresponding different functions. This study provides new insight into the biological features and clinical use of MSCs at the single-cell level, which may contribute to expanding their application in the clinic.

Summary Mucosal-associated invariant T (MAIT) cells are innate-like unconventional T cells that are abundant in humans and have attracted increasing attention in recent years. Mesenchymal stem cells (MSCs) are crucial regulators of immune cells. However, whether MAIT cells are regulated by MSCs is unclear. Here, we explored the effect of MSCs on MAIT cells and revealed the underlying mechanism. We found that MSCs did not influence the proliferation of MAIT cells but strikingly induced an activated phenotype with an increased expression of CD69, TNF-α, IFN-γ, and granzyme B. Moreover, MSCs activated MAIT cells in a TCR-MR1-independent mechanism through MSC-secreted IL-15. We revealed that MSC-derived IL-15 activated MAIT cells by enhancing autophagy activity, which was abolished by the autophagy inhibitor 3-methyladenine. Based on our findings, MAIT cells are activated by MSCs through IL-15-induced autophagy, which may help elucidate the mechanisms underlying some immune responses and diseases and provide guidance for future research., Highlights • MSCs activate MAIT cells to express higher levels of TNF-α, IFN-γ, and granzyme B • MAIT cells are activated in a TCR-MR1-independent way through MSC-secreted IL-15 • MSC-derived IL-15 activates MAIT cells by enhancing autophagy activity, In this article, Shen Huiyong and colleagues show that MSCs do not influence the proliferation of MAIT cells but strikingly induce an activated phenotype with an increased expression of CD69, TNF-α, IFN-γ, and granzyme B. Mechanistically, MSC-derived IL-15 enhances the autophagy level of MAIT cells and results in MAIT cell activation.

An imbalance of human mesenchymal stem cells (hMSCs) adipogenic and osteogenic differentiation is crucial in the pathogenesis of osteoporosis, and elucidation of the underlying mechanism is urgently needed. APPL1, an adaptor protein of the adiponectin receptor, was recently shown to be closely related to bone mass. However, the role of APPL1 in the imbalance of hMSC differentiation in osteoporosis is unclear. Therefore, we aimed to explore the mechanisms by which APPL1 alters hMSCs adipogenic differentiation in osteoporosis. Here, we found that APPL1 expression was downregulated in elderly patients with osteoporosis and in mouse osteoporosis model. APPL1 negatively regulated hMSC adipogenic differentiation in vivo and in vitro. Mechanistically, by enhancing ubiquitination-mediated Myoferlin degradation, downregulated APPL1 expression increased the risk of lysosome dysfunction during hMSCs adipogenic differentiation. Lysosomal dysfunction inhibited autophagy flux by suppressing autophagosome degradation and promoted hMSC differentiation towards the adipocyte lineage. Our findings suggest that APPL1/Myoferlin downregulation promoted hMSCs adipogenic differentiation by inhibiting autophagy flux, further impairing the balance of hMSCs adipogenic and osteogenic differentiation in osteoporosis; the APPL1/ Myoferlin axis may be a promising diagnostic and therapeutic target for osteoporosis.

Objective To investigate risk factors of cage retropulsion after posterior lumbar interbody fusion (PLIF) in China and to establish a scoring system of cage retropulsion. Methods The retrospective analysis was based on two hospital databases. The medical data records of posterior lumbar interbody fusion with cage retropulsion were selected from August 2009 to August 2019. Inclusion and exclusion criteria were set in advance. Risk factors including patients' baseline demographics (age, gender, operation diagnosis time difference), preoperative neurological symptoms, whether the fusion involves single or double segments, screw type, intraoperative compression, preoperative bone mineral density, whether there are neurological symptoms before surgery, whether there is urine dysfunction before surgery, disease type, complete removal of the endplate, and patient's education level. The research endpoint was the retropulsion of fusion cages. The Kaplan–Meier (K‐M) method was used to analyze potential risk factors, and multivariate Cox regression was used to identify independent risk factors (P, Time of cage retropulsion in different risk groups (days). According to intraoperative pressure and the complete removal of intraoperative endplate, we can evaluate patients with posterior lumbar fusion to determine the expected time of cage displacement in order to guide the postoperative prevention and follow‐up treatment

Objective To compare the clinical and radiographic outcomes between the Tri‐Lock Bone Preservation Stem (BPS) and the conventional standard Corail stem in primary total hip arthroplasty (THA). Methods From March 2012 to May 2014, we retrospectively reviewed 84 patients (104 hips) who received Tri‐Lock (BPS) and 84 patients (115 hips) who received conventional standard Corail stem in THA. Their mean ages were 53.12 ± 2.32 years and 52.00 ± 2.11 years, respectively. The clinical outcomes were assessed by Western Ontario and McMaster University Osteoarthritis Index (WOMAC), Pain Visual Analogue Scale (VAS) and Harris Hip Score (HHS). The radiological outcomes were evaluated by the radiological examination. Accordingly, Intraoperative and postoperative complications were observed as well. Results The mean follow‐up time was 48.23 ± 2.91 months in the Tri‐Lock (BPS) group and 49.11 ± 2.11 months in the Corail group, respectively. The bleeding volumes in two groups were comparable (169.22 ± 58.11 mL vs 179.30 ± 59.14 mL, P = 0.003), with more bleeding volume in Corail group patients, while no statistically significance with respect to operation time was observed (65.41 ± 6.24 min vs 63.99 ± 6.33 min, P = 0.567). The rates of intraoperative fracture was 8% for the Corail group while 1% for the Tri‐Lock (BPS) group (8% vs 1%, P = 0.030). At final follow‐up, no statistical differences in regard to HHS, WOMAC, and Pain VAS were revealed between the two groups (P > 0.05). The rate of thigh pain was higher in Corail group than in Tri‐lock (BPS) group (5% vs 0%, P = 0.043). However, incidence of stress shielding in grade 1 was higher in Tri‐Lock (BPS) than in the Corail group (76% vs 23%, P, This study demonstrated that the Tri‐lock (BPS) has similar performances in clinic outcomes compared to the Corail stem. Furthermore, the Tri‐lock (BPS) stem has lower incidence of thigh pain, stress shielding and intra‐operative fracture. In addition, taking into account of patients factors, including bone deficiency and convenience of extraction of the stem in hip revision, we recommend the Tri‐lock (BPS) stem may be a good alternative.

Dysregulated angiogenesis of mesenchymal stem cells (MSCs) is closely related to inflammation and disrupted bone metabolism in patients with various autoimmune diseases. However, the role of MSCs in the development of abnormal angiogenesis in patients with ankylosing spondylitis (AS) remains unclear. In this study, we cultured human umbilical vein endothelial cells (HUVECs) with bone marrow‐derived MSCs from patients with AS (ASMSCs) or healthy donors (HDMSCs) in vitro. Then, the cocultured HUVECs were assayed using a cell counting kit‐8 (CCK‐8) to evaluate the cell proliferation. A wound healing assay was performed to investigate cell migration, and a tube formation assay was conducted to determine the angiogenesis efficiency. ASMSCs exhibited increased angiogenesis, and increased expression of SMAD‐specific E3 ubiquitin ligase 2 (Smurf2) in MSCs was the main cause of abnormal angiogenesis in patients with AS. Downregulation of Smurf2 in ASMSCs blocked angiogenesis, whereas overexpression of Smurf2 in HDMSCs promoted angiogenesis. The pro‐angiogenic effect of Smurf2 was confirmed by the results of a Matrigel plug assay in vivo. By functioning as an E3 ubiquitin ligase in MSCs, Smurf2 regulated the levels of pentraxin 3 (PTX3), which has been shown to suppress angiogenesis through the PTX3‐fibroblast growth factor 2 pathway. Moreover, Smurf2 transcription was regulated by activating transcription factor 4‐induced endoplasmic reticulum stress. In conclusion, these results identify novel roles of Smurf2 in negatively regulating PTX3 stability and promoting angiogenesis in ASMSCs., SMAD‐specific E3 ubiquitin protein ligase 2 (Smurf2) transcription is regulated by activating transcription factor 4‐induced endoplasmic reticulum stress, and Smurf2 negatively regulates pentraxin 3 (PTX3) stability in ankylosing spondylitis mesenchymal stem cells and abnormally promotes angiogenesis through the PTX3‐fibroblast growth factor 2 axis.

To introduce an all-inside modified Broström technique to suture the anterior talofibular ligament (ATFL) and inferior extensor retinaculum (IER) under arthroscopy and to compare its outcomes with those of the conventional open procedure.All patients who underwent arthroscopic or open repair of the ATFL between June 2014 and December 2017 were included in this study. Visual analog scale (VAS), Karlsson and Peterson (K-P), American Orthopedic Foot and Ankle Society (AOFAS) ankle/hindfoot, and Tegner activity scores, as well as manual anterior drawer test (ADT), were used to evaluate the patients preoperatively and ≥2 years after surgery. The Sefton grading system was used to assess the level of satisfaction after surgery. Detailed surgical data and intraoperative findings were documented at the time of surgery.A total of 67 patients, 31 in the arthroscopic group and 36 in the open group, were included in this study (43 men and 24 women, mean body mass index 24.00, range 19.53 to 30.03). The surgical duration in the arthroscopic group (median, 34 minutes; range, 25 to 74) was significantly shorter than that in the open group (mean, 43.08 ± 8.11 minutes; 95% confidence interval [CI] 40.34 to 45.83) (P = .007). At the last follow-up, the subjective functional scores and ADT results improved significantly in both cohorts (P.001). However, no significant difference was found in the VAS score (1.74 ± 1.24, 95% CI 1.29 to 2.2, in the open group versus 1.58 ± 1.2, 95% CI 1.18 to 1.99, in the arthroscopic group; P = .581), AOFAS score (91.71 ± 5.46, 95% CI 89.71 to 93.71, versus 90.67 ± 5.59, 95% CI 88.78 to 92.56; P = .444), K-P score (87.52 ± 7.59, 95% CI 84.73 to 90.3, versus 88.75 ± 5.56, 95% CI 86.87 to 90.63; P = .446), and ADT evaluation (normal: 96.77% versus 94.44%, P = .557) between the arthroscopic and open groups, respectively. In addition, 28 cases (90.32%) in the arthroscopic group and 32 (88.89%) in the open group achieved satisfactory results based on the Sefton grading system (P = .736). Seventeen patients (47.2%) in the open group and 18 patients (58.1%) in the arthroscopic group underwent Tegner evaluation after surgery, which showed no significant difference (5, interquartile range [IQR] 1 in the open group versus 5, IQR 3 in the arthroscopic group; P = .883). Complications were reported in 4 (11.1%) and 2 (6.5%) patients who underwent open and arthroscopic surgeries, respectively (P = .813).Both open and arthroscopic modified Broström surgeries generated favorable outcomes, with a significant improvement compared with the preoperative condition. Compared with the open Broström-Gould procedure, the all-inside arthroscopic modified Broström technique produced equivalent functional and clinical results at a minimum of 2 years after the operation, with a shorter surgical duration. Arthroscopic repair might be a safe and viable alternative to open surgery for lateral ankle stabilization.III.

Rationale: Osteosarcoma (OS), the most common type of bone tumor, which seriously affects the patients' limb function and life quality. OS has a strong tendency of lung metastasis, and the five-year survival rate of patients with metastatic osteosarcoma is less than 20%. Thus, new treatment targets and strategies are urgently needed. Methods: The expression of the histone demethylase KDM6B and H3K27me3 levels in OS specimens were analyzed using quantitative PCR and immunohistochemical assays. The biological functions of KDM6B were determined using in vitro transwell, wound healing assays, and an in vivo orthotopic injection-induced lung metastasis model. Subsequently, chromatin immunoprecipitation sequencing (ChIP-seq) combined with transcriptomic RNA sequencing (RNA-seq), and subsequent ChIP-qPCR, western blot, and aerobic glycolysis assays were used to explore the mechanism of KDM6B function and validate the candidate target gene of KDM6B. Results: KDM6B expression was significantly upregulated in OS patients, and high KDM6B expression was associated with poorer prognosis in OS patients. Targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo. RNA-seq and ChIP-seq analysis revealed that KDM6B increases lactate dehydrogenase LDHA expression in OS cells by directly mediating H3K27me3 demethylation. The phenotypes of inhibited cell metastasis in KDM6B-knockdown OS cells was reversed upon overexpression of LDHA. Finally, a small molecule inhibitor targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo. Conclusions: Collectively, we elucidated that upregulated KDM6B facilitates tumor metastasis in OS via modulating LDHA expression. Our findings deepen the recognition of OS metastasis mechanism and suggest that KDM6B might be a new potential therapeutic target for the treatment of OS (especially highly metastatic OS).

BackgroundThe zinc transporters Zrt- and Irt-related protein (ZIP/SLC39) are overexpressed in human tumors and correlate with poor prognosis; however, their contributions to carcinogenesis and chemoresistance in osteosarcoma (OS) remain unclear.MethodsWe collected 64 OS patient tissues with (n = 12) or without (n = 52) chemotherapy. The expression levels of ZIP10 were measured by immunohistochemistry and applied to prognostic analysis. ZIP10 was knocked down or overexpressed in OS cell lines to explore its effect on proliferation and chemoresistance. RNA sequencing, quantitative real-time PCR, and western blotting analysis were performed to explore ZIP10-regulated downstream target genes. A xenograft mouse model was established to evaluate the mechanisms by which ZIP10 modulates chemoresistance in OS cells.ResultsThe expression of ZIP10 was significantly induced by chemotherapy and highly associated with the clinical outcomes of OS. Knockdown of ZIP10 suppressed OS cell proliferation and chemoresistance. In addition, ZIP10 promoted Zn content-induced cAMP-response element binding protein (CREB) phosphorylation and activation, which are required for integrin α10 (ITGA10) transcription and ITGA10-mediated PI3K/AKT pathway activation. Importantly, ITGA10 stimulated PI3K/AKT signaling but not the classical FAK or SRC pathway. Moreover, overexpression of ZIP10 promoted ITGA10 expression and conferred chemoresistance. Treatment with the CREB inhibitor 666–15 or the PI3K/AKT inhibitor GSK690693 impaired tumor chemoresistance in ZIP10-overexpressing cells. Finally, a xenograft mouse model established by subcutaneous injection of 143B cells confirmed that ZIP10 mediates chemotherapy resistance in OS cells via the ZIP10-ITGA10-PI3K/AKT axis.ConclusionsWe demonstrate that ZIP10 drives OS proliferation and chemoresistance through ITGA10-mediated activation of the PI3K/AKT pathway, which might serve as a target for OS treatment.

TNF receptor-associated factor 4 (TRAF4), a member of the TRAF family, plays an important role in the embryogenesis and development of the bone system. Mesenchymal stem cells (MSCs), which are the primary origin of osteoblasts in vivo, are key cells in bone development; however, whether TRAF4 modulates the osteogenic capacity of MSCs has never been explored. In this study, we demonstrated that TRAF4 positively regulates the osteogenic process of MSCs both in vitro and in vivo. In addition, we further demonstrated that TRAF4 modulates the osteogenic process of MSCs by acting as an E3 ubiquitin ligase to mediate the K48-linked ubiquitination of Smurf2 at the K119 site and cause degradation. Furthermore, TRAF4 was abnormally decreased in bone sections of ovariectomized rat and osteoporosis patients. Taken together, our findings suggest that TRAF4 positively regulates the osteogenic differentiation of MSCs by acting as an E3 ubiquitin ligase to degrade Smurf2. These results emphasize the critical role of TRAF4 in bone formation and could not only improve the clinical use of MSCs in tissue engineering but also clarify the pathogenesis of bone metabolism disorders.

The mechanism of pathological osteogenesis in Ankylosing spondylitis (AS) is largely unknown. Our previous studies demonstrated that the imbalance between BMP-2 and Noggin secretion induces abnormal osteogenic differentiation of marrow-derived mesenchymal stem cells (MSCs) from AS patients in a two-dimensional culture environment. In this study, HA/β-TCP scaffolds were further used as a three-dimensional (3D) biomimetic culture system to mimic the bone microenvironment in vivo to determine the abnormal osteogenic differentiation of AS-MSCs. We demonstrated that when cultured in HA/β-TCP scaffolds, AS-MSCs had a stronger osteogenic differentiation capacity than that of MSCs from healthy donors (HD-MSCs) in vitro and in vivo. This dysfunction resulted from BMP2 overexpression in AS-MSCs, which excessively activated the Smad1/5/8 and ERK signalling pathways and finally led to enhanced osteogenic differentiation. Both the signalling pathway inhibitors and siRNAs inhibiting BMP2 expression could rectify the enhanced osteogenic differentiation of AS-MSCs. Furthermore, BMP2 expression in ossifying entheses was significantly higher in AS patients. In summary, our study demonstrated that AS-MSCs possess enhanced osteogenic differentiation in HA/β-TCP scaffolds as a 3D biomimetic microenvironment because of BMP2 overexpression, but not Noggin. These results provide insights into the mechanism of pathological osteogenesis, which can aid in the development of niche-targeting medications for AS.

N6-methyladenosine (m6A) modification is widespread in messenger RNAs and increasing evidence suggests the crucial roles of m6A in cell differentiation and tissue development. However, whether m6A modulates the osteogenic differentiation of mesenchymal stem cells (MSCs) has not been fully elucidated. Here we show that conditional knockout of the demethylase Alkbh5 in bone marrow MSCs strengthened bone mass in mice. Loss- and gain-of-function studies demonstrated that ALKBH5 negatively regulates the osteogenic differentiation of MSCs in vitro. At a mechanistic level, meRIP-seq and RNA-seq in MSCs following knockdown of ALKBH5 revealed changes in transcripts of PRMT6 containing consensus m6A motifs required for demethylation by ALKBH5. Furthermore, we found that ALKBH5 accelerates the degradation rate of PRMT6 mRNA in an m6A-dependent manner, and that the ALKBH5-PRMT6 axis regulates the osteogenesis of MSCs, mainly through activation of the PI3K/AKT pathway. Thus, our work reveals a different facet of the novel ALKBH5-PRMT6 axis that modulates the osteogenic differentiation of MSCs, which can serve as a target to improve the clinical use of MSCs.

Background Ankylosing spondylitis is a progressive, disabling joint disease that affects millions worldwide. Given its unclear etiology, studies of ankylosing spondylitis relied heavily on drug-induced or transgenic rodent models which retain only partial clinical features. There is obviously a lack of a useful disease model to conduct comprehensive mechanistic studies. Methods We followed a group of cynomolgus monkeys having joint lesions reported of spinal stiffness for 2 years by conducting hematological testing, radiographic examination, family aggregation analysis, pathological analysis, and genetic testing. Results The results confirmed that these diseased animals suffered from spontaneous ankylosing spondylitis with clinical features recapitulating human ankylosing spondylitis disease progression, manifested by pathological changes and biochemical indicators similar to that of ankylosing spondylitis patients. Conclusion The study offers a promising non-human primate model for spontaneous ankylosing spondylitis which may serve as an excellent substitute for its pre-clinical research.

OBJECTIVE: To evaluate the effectiveness of cement-injectable cannulated pedicle screw combined with multiple level Schwab grade Ⅰ osteotomy for chronic thoracolumbar osteoporotic fractures with kyphosis. METHODS: The clinical data of 27 patients with symptomatic chronic thoracolumbar osteoporotic fractures combined with kyphosis treated between June 2015 and June 2017 were retrospectively analysed. Among them, there were 8 males and 19 females, with an average age of 69.5 years (range, 56-81 years). The damage segment (kyphosis vertex) included T(11) in 4 cases, T(12) in 12 cases, L(1) in 10 cases, and L(2) in 1 case. The disease duration ranged from 3 to 21 months, with an average of 12.5 months. The T value of lumbar vertebral bone mineral density ranged from −4.9 to −2.5, with an average value of −3.61. The American Spinal Injury Association (ASIA) classification was used to evaluate spinal cord injury, there were 1 case of grade D and 26 cases of grade E. The visual analogue scale (VAS) score, Oswestry disability index (ODI), kyphosis Cobb angle of fracture site, and sagittal vertical axis (SVA) data were obtained before operation, at 2 weeks after operation, 3 months after operation, and last follow-up, to evaluate the quality of life and improvement of sagittal spine parameters. RESULTS: No complications related to pedicle screw and bone cement occurred. The incisions healed by first intention in 26 cases, and 1 incision healed after dressing change due to poor blood glucose control. There were no complications such as bedsore, hypostatic pneumonia, or deep venous thrombosis. All patients were followed up 8-24 months, with an average of 16.6 months. The VAS score, ODI score, Cobb angle, and SVA were significantly improved when compared with those before operation (P0.05); the VAS score and ODI score at 3 months after operation and last follow-up were significantly better than those at 2 weeks after operation (P0.05); SVA at last follow-up was significantly worse than that at 2 weeks and 3 months after operation (P0.05). During the follow-up period, there was no complication such as pedicle screw loosening, breakage or cutting, adjacent vertebral fracture, proximal junctional kyphosis, and so on. CONCLUSION: For the chronic thoracolumbar osteoporotic fractures combined with kyphosis, the cement-injectable cannulated pedicle screw and multiple level Schwab grade Ⅰ osteotomy has the advantages of less operation trauma, quick recovery, and remarkable effectiveness.

BACKGROUND To introduce a novel robotic system 'Orthbot' that has been developed and tested as a surgical assistant for auto-placement of the K-wire in lumbar fusion. METHODS This is a multi-centre, randomized controlled clinical study that includes 56 patients (robot group, RG: 27, free-hand group, FG: 29). Following the pre-operative planning and intra-operative fluoroscopic images, the 'Orthbot' automatically completed registration and K-wire placement under the supervision of the surgeon. Deviation distance (DD) and deviation angle (DA) were used as the primary parameters to evaluate the accuracy of the robotic system. RESULTS The average DD was 0.95 ± 0.377 mm and 4.35 ± 2.01 mm, respectively in the RG and FG (p

Ankylosing spondylitis (AS) is a rheumatic disease with pathological osteogenesis that causes bony ankylosis and even deformity over time. Mesenchymal stem cells (MSCs) are multipotent stem cells that are the main source of osteoblasts. We previously demonstrated that enhanced osteogenic differentiation of MSCs from AS patients (ASMSCs) is related to pathological osteogenesis in AS. However, the more concrete mechanism needs further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development. Single-nucleotide polymorphisms (SNPs) regulate the formation and interaction of SEs and denote genes accounting for AS susceptibility. Via integrative analysis of multiomic data, including histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation sequencing (ChIP-seq), SNPs and RNA sequencing (RNA-seq) data, we discovered a transcription network mediated by AS SNP-adjacent SEs (SASEs) in ASMSCs and identified key genes, such as Toll-like receptor 4 (TLR4), interleukin 18 receptor 1 (IL18R1), insulin-like growth factor binding protein 4 (IGFBP4), transportin 1 (TNPO1) and proprotein convertase subtilisin/kexin type 5 (PCSK5), which are pivotal in osteogenesis and AS pathogenesis. The SASE-regulated network modulates the enhanced osteogenic differentiation of ASMSCs by synergistically activating the PI3K-Akt, NF-kappaB and Hippo signaling pathways. Our results emphasize the crucial role of the SASE-regulated network in pathological osteogenesis in AS, and the preferential inhibition of ASMSC osteogenic differentiation by JQ1 indicates that SEs may be attractive targets in future treatment for new bone formation in AS.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic OPLL. To date, the specific molecular pathogenesis of OPLL has not been clearly elucidated. In this study, bone marrow-derived mesenchymal stem cells obtained from healthy donors (HD-MSCs) and patients with OPLL (OPLL-MSCs) were cultured in osteogenic differentiation medium for 21 days. The osteogenic differentiation capacity was determined by alizarin red S (ARS) and alkaline phosphatase (ALP) assays. Gene expression levels of osteoblastic markers were measured by quantitative reverse transcription-polymerase chain reaction. Protein levels of related genes and the activation of related signaling pathways were measured by western blotting. LDN193189 was used to inhibit the Smad1/5/8 pathway, and small interfering RNA was used to regulate BMP2 expression. Our results showed that the OPLL-MSCs had stronger ARS staining and ALP activity and higher expression of RUNX2, Osterix, and OCN than the HD-MSCs. During osteogenic differentiation, the Smad1/5/8 pathway was overactivated in the OPLL-MSCs, and LDN193189 inhibition reversed the enhanced osteogenic ability of these cells. Besides, BMP2 was upregulated in the OPLL-MSCs. After BMP2 knockdown, the abnormal osteogenic differentiation of OPLL-MSCs was rescued. Thus, abnormal activation of the BMP2-Smad1/5/8 pathway induces enhanced osteogenic differentiation of OPLL-MSCs compared with HD-MSCs. These findings reveal a mechanism of pathological osteogenesis in OPLL and provide a new perspective on inhibiting pathological osteogenesis by regulating BMP2.

Osteoporosis is a common systemic skeletal disorder resulting in bone fragility and increased fracture risk. It is still necessary to explore its detailed mechanisms and identify novel targets for the treatment of osteoporosis. Previously, we found that a lncRNA named GAS5 in human could negatively regulate the lipoblast/adipocyte differentiation. However, it is still unclear whether GAS5 affects osteoblast differentiation and whether GAS5 is associated with osteoporosis. Our current research found that GAS5 was decreased in the bones and BMSCs, a major origin of osteoblast, of osteoporosis patients. Mechanistically, GAS5 promotes the osteoblast differentiation by interacting with UPF1 to degrade SMAD7 mRNA. Moreover, a decreased bone mass and impaired bone repair ability were observed in Gas5 heterozygous mice, manifesting in osteoporosis. The systemic supplement of Gas5-overexpressing adenoviruses significantly ameliorated bone loss in an osteoporosis mouse model. In conclusion, GAS5 promotes osteoblast differentiation by targeting the UPF1/SMAD7 axis and protects against osteoporosis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease possessing a morbid serum microenvironment with enhanced oxidative stress. Long-term exposure to an oxidative environment usually results in cellular senescence alone with cellular dysfunction. Mesenchymal stem cells (MSCs) are a kind of stem cell possessing strong capabilities for immunoregulation, and senescent MSCs may increase inflammation and participate in AS pathogenesis. The objective of this study was to explore whether and how the oxidative serum environment of AS induces MSC senescence. Here, we found that AS serum facilitated senescence of MSCs in vitro, and articular tissues from AS patients exhibited higher expression levels of the cell cycle arrest-related proteins p53, p21 and p16. Importantly, the levels of advanced oxidative protein products (AOPPs), markers of oxidative stress, were increased in AS serum and positively correlated with the extent of MSC senescence induced by AS serum. Furthermore, MSCs cultured with AS serum showed decreased mitochondrial membrane potential and ATP production together with a reduced oxygen consumption rate. Finally, we discovered that AS serum-induced mitochondrial dysfunction resulted in elevated reactive oxygen species (ROS) in MSCs, and ROS inhibition successfully rescued MSCs from senescence. In conclusion, our data demonstrated that the oxidative serum environment of AS facilitated MSC senescence through inducing mitochondrial dysfunction and excessive ROS production. These results may help elucidate the pathogenesis of AS and provide potential targets for AS treatment.

Ankylosing spondylitis (AS) is a type of rheumatic disease characterized by chronic inflammation and pathological osteogenesis in the entheses. Previously, we demonstrated that enhanced osteogenic differentiation of MSC from AS patients (AS-MSC) resulted in pathological osteogenesis, and that during the enhanced osteogenic differentiation course, AS-MSC induced TNF-α-mediated local inflammation. However, whether TNF-α in turn affects AS-MSC remains unknown. Herein, we further demonstrate that a high-concentration TNF-α treatment triggers enhanced directional migration of AS-MSC in vitro and in vivo, which enforces AS pathogenesis. Mechanistically, TNF-α leads to increased expression of ELMO1 in AS-MSC, which is mediated by a METTL14 dependent m

Background A few months ago, the Bioscience Reports journal showed that growth differentiation factor 5 (GDF5) rs143383 genetic polymorphism increases the susceptibility of knee osteoarthritis (KOA), but previous studies’ results have debates about available data. Considering the availability of more recent data, we focus on clarifying the relationship of KOA and GDF5 rs143383 genetic polymorphism by a meta-analysis of case-control trial data. Methods The eligible studies from the time of database established to Oct. 2019 were collected from PubMed, Springer, Cochrane library, Web of Science, China National Knowledge Infrastructure (CNKI), and Wan Fang library. Odds ratios (OR) and 95% confidence intervals (CI) were used to estimate the association between these polymorphisms and KOA risk. The meta-analysis was completed by STATA 18.0 software. Results A total of 196 studies were collected, 16 of them included in final meta-analysis (7997 cases and 12,684 controls). There was significant association between GDF5 rs143383 polymorphism and KOA in all genetic models (for Allele model (C versus T): OR = 0.84 (95% CI = 0.76–0.91); dominate model (CC+CT versus TT): OR = 0.80 (95% CI = 0.72–0.90); recessive model (CC versus CT+TT): OR = 0.79 (95% CI = 0.68–0.92); heterozygote model (CT versus CC+TT): OR = 0.89 (95% CI = 0.80–0.97); homozygous model (CC versus TT): OR = 0.71 (95% CI = 0.60–0.85)). In the subgroup analysis, we obtained the results that there is no significance among Asians. Conclusion GDF5 rs143383 genetic polymorphism increases the risk of KOA among Caucasians; CC genotype and C allele are protective factors for the susceptibility of KOA among Caucasians.

ObjectiveTo explore the roles of human mesenchymal stem cell (hMSC) death-associated protein kinase 1 (DAPK1) in modulating CD4+ T lymphocyte proliferation.MethodsHuman MSCs and peripheral blood mononuclear cells were isolated and cocultured in vitro for 3 days. Lentiviral-mediated RNA interference (LV-sh-DAPK1) was used to silence DAPK1 expression in hMSCs. Expression of DAPK1 was assessed by western blotting. Transcriptional levels of DAPK1, transforming growth factor-β1, indoleamine 2,3-dioxygenase, inducible nitric oxide synthase, interleukin (IL)-6, suppressor of cytokine signaling 1, IL-10 and cyclooxygenase-2 were investigated by quantitative PCR. Levels of IL-10 were assessed by ELISA. Proliferation of CD4+ T cells was assessed by flow cytometry.ResultsDAPK1 was abundantly expressed in ex vivo-expanded hMSCs and expression was positively correlated with hMSC suppression of CD4+ T cell proliferation. Silencing of DAPK1 in hMSCs reduced the ability of these cells to inhibit CD4+ T cell proliferation and resulted in decreased IL-10 levels compared with untreated controls. Exogenous supplementation with recombinant human IL-10 in DAPK1-silenced hMSCs restored immunosuppression of CD4+ T cells.ConclusionsThe DAPK1-IL-10 axis mediates a novel immunoregulatory function of hMSCs toward CD4+ T cells.

Ankylosing spondylitis (AS) is a common inflammatory autoimmune disease, characterized by pathological osteogenesis. Mesenchymal stem cells (MSCs), as the main source of osteoblasts, participate in bone remodeling not only through differentiation into osteoblasts but also through indirect regulation of osteoclastogenesis. Our previous study indicated that the stronger osteogenic differentiation of MSCs from AS patients (ASMSCs) involved in pathological osteogenesis. However, whether there is any abnormality in the regulation of osteoclastogenesis by ASMSCs remains unclear. In this study, ASMSCs or MSCs from healthy donors (HDMSCs) were co-cultured with CD14 + monocytes in osteoclast induction medium. Our results demonstrated that ASMSCs exhibited a stronger capacity to inhibit osteoclastogenesis than HDMSCs. To explore underlying mechanisms, cytokine array assays were performed, showing that ASMSCs secreted more CXCL5 than HDMSCs, which was confirmed by enzyme-linked immunosorbent assays. Moreover, inhibition of osteoclastogenesis by ASMSCs was recovered by decreasing CXCL5. Besides, the inhibitory effect of CXCL5 on osteoclastogenesis was confirmed by exogenous addition. Bioinformatics analysis was applied to find the interaction between miR-4284 and CXCL5, which was verified by luciferase reporter assays. Furthermore, we used miR-4284 inhibitors or mimics to prove that the expression of CXCL5 was regulated by miR-4284. Further analysis showed that downregulation of miR-4284 in MSCs resulted in increase of CXCL5, markedly inhibiting osteoclastogenesis, whereas upregulation of miR-4284 in MSCs had the opposite effect. Our findings indicate that ASMSCs exhibit a stronger capacity to inhibit osteoclastogenesis than HDMSCs through the miR-4284/CXCL5 axis, which provide a new perspective on the mechanism of pathologic osteogenesis in AS.

Background: Mesenchymal stem cells (MSCs) selectively differentiate into adipocytes or osteoblasts, and several molecules control the fate determination of MSCs. Understanding these key checkpoints greatly contributes to the ability to induce specific MSC differentiation for clinical applications. In this study, we aimed to explore whether TNF receptor-associated factor 4 (TRAF4) affects MSC adipogenic differentiation, which we previously reported that could positively regulated the osteogenic differentiation. Methods: Western blotting and Real-time Polymerase Chain Reaction were used to detected the expression pattern of TRAF4 during adipogenic differentiation. Lentivirus was constructed to regulate TRAF4 expression, and oil red O staining and Western blotting were used to assess its role in adipogenesis, which was confirmed in vivo by implanting an MSC-matrigel mixture into nude mice. Western blotting was used to detect the activated signaling pathways, and a specific inhibitor and agonist were used to clear the roles of the key signaling pathways. Additionaly, Co-Immunoprecipitation was conducted to find that Pyruvate kinase isozyme type M2 (PKM2) interacts with TRAF4, and to further explore their binding and functional domains. Finally, an RNA-binding protein immunoprecipitation assay and Western blotting were used to detect whether N6-methyladenosine mediates the decreased TRAF4 expression during adipogenic differentiation. Findings: The results demonstrated that TRAF4 negatively regulates MSC adipogenesis in vitro and in vivo. Mechanistically, we revealed that TRAF4 binds to PKM2 to activate the kinase activity of PKM2, which subsequently activates β-catenin signaling and then inhibits adipogenesis. Furthermore, TRAF4 downregulation during adipogenesis is regulated by ALKBH5-mediated N6-methyladenosine RNA demethylation. Interpretation: TRAF4 negatively regulates the adipogenesis of MSCs by activating PKM2 kinase activity, which may act as a checkpoint to fine-tune the balance of adipo-osteogenic differentiation, and suggests that TRAF4 may be a novel target of MSCs in clinical use and may also illuminate the underlying mechanisms of bone metabolic diseases. Funding: This study was supported by the National Natural Science Foundation of China (81871750 and 81971518) and the Science and Technology Project of Guangdong Province (2019B02023600 and 2017A020215070). Keywords: TRAF4, Mesenchymal stem cells, Adipogenic differentiation, PKM2

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) are the main source of osteoblasts in vivo and are widely used in stem cell therapy. Previously, we analyzed long noncoding RNA (lncRNA) expression profiles during BM‐MSC osteogenesis, and further investigation is needed to elucidate how lncRNAs regulate BM‐MSC osteogenesis. Herein, we used customized microarrays to determine lncRNA expression profiles in BM‐MSCs on days 0 and 10 of osteogenic differentiation. In addition, we identified a novel osteogenesis‐associated lncRNA (lncRNA‐OG) that is upregulated during this process. Functional assays showed that lncRNA‐OG significantly promotes BM‐MSC osteogenesis. Mechanistically, lncRNA‐OG interacts with heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein to regulate bone morphogenetic protein signaling pathway activation. Surprisingly, hnRNPK positively regulates lncRNA‐OG transcriptional activity by promoting H3K27 acetylation of the lncRNA‐OG promoter. Therefore, our study revealed a novel lncRNA with a positive function on BM‐MSC osteogenic differentiation and proposed a new interaction between hnRNPK and lncRNA. stem cells 2018 Stem Cells 2019;37:270–283, (A): During the osteogenesis of bone marrow‐derived mesenchymal stem cells, osteogenesis‐associated long noncoding RNA interacts with hnRNPK to promote the expression of bone morphogenetic proteins family, which ultimately promotes the process of osteogenesis. (B): Interestingly, hnRNPK can also bind to the promoter of osteogenesis‐associated long noncoding RNA, promoting osteogenesis‐associated long noncoding RNA transcriptional activity by increasing histone H3 lysine 27 acetylation (H3K27ac).

The aim of this study is to evaluate the influences of different bone graft heights on the size of the intervertebral foramen, which will help determine the optimal graft height in clinical practice. Six fresh adult cadavers were used, with the C5-C6 vertebral column segment defined as the functional spinal unit (FSU). After discectomy, the C5/6 intervertebral height was set as the baseline height (normal disc height). We initially used spiral computed tomography (CT) to scan and measure the middle area of the intervertebral foramen when at the baseline height. Data regarding the spatial relationship of C5-C6 were subsequently collected with a laser scanner. Grafting with four different sized grafts, namely, grafts of 100, 130, 160, and 190% of the baseline height, was implanted. Moreover, we scanned to display the FSU in the four different states using Geomagic8.0 studio software. Multiple planar dynamic measurements (MPDM) were adopted to measure the intervertebral foramen volume, middle area, and areas of internal and external opening. MPDM with a laser scanner precisely measured the middle area of the intervertebral foramen as spiral CT, and it is easy to simulate the different grafts implanted. With the increase of the bone graft height, the size of the intervertebral foramen began to decrease after it increased to a certain point, when grafts of 160% of the baseline height implanted. MPDM of the intervertebral foramens with laser scanning three-dimensional (3D) reconstitution are relatively objective and accurate. The recommended optimal graft height of cervical spondylosis is 160% of the mean height of adjacent normal intervertebral spaces.

DEP domain containing 1 (DEPDC1) is recently reported to be overexpressed in several types of human cancer; however the role of DEPDC1 in prostate cancer remains to be investigated. Herein, we identified that the DEPDC1 mRNA and protein expression levels were dramatically increased in prostate cancer tissues and cell lines. Overexpression of DEPDC1 promoted, but depletion of DEPDC1 inhibited cell proliferation by regulating the G1-S phase cell cycle transition. Importantly, we found that DEPDC1 was essential for the tumor growth and formation of bone metastases of prostate cancer cells in vivo. Finally, we demonstrated that DEPDC1 interacted with E2F1 and increased its transcriptional activity, leading to hyper-activation of E2F signaling in prostate cancer cells. Our findings reveal an oncogenic role of DEPDC1 in prostate cancer progression via activation of E2F signaling, and suggest DEPDC1 might be a potential therapeutic target against the disease.

Macrophage-related inflammatory response is one of the main biological factors resulting in failure of anterior cruciate ligament (ACL) reconstruction, although the specific pathomechanism remains to be clarified. Our aim was to investigate the association between graft loosening and macrophage-related inflammation in cases of loosening of reconstructed ACL autografts. Tissue samples were obtained from 21 patients who underwent a second-look arthroscopy within the first year after arthroscopic ACL reconstruction using single-bundle hamstring tendon autografts. Possible biological factors of graft loosening were analyzed using polymerase chain reaction, Western blot, and hematoxylin/eosin and immunohistochemical staining of graft tissue samples obtained during the second-look arthroscopy. Graft loosening was closely related to increased gene and protein expression of inflammatory cytokines (TNF-α, IL-6, and IL-8) and activation of the inflammation-related toll-like receptor (TLR) signaling (TLR2 and TLR4). The molecular expression of TGF-β and type I and III collagen was also inhibited to varying degrees, with decreased vascularization of the graft due to an inhibition of VEGF. iNOS, a marker of M1 macrophage activation, was highly expressed in cases of graft loosening, with no effect of M2 macrophages identified. The activation of M1 macrophages and aseptic inflammation signaling is an important biological factor of graft loosening after ACL reconstruction, affecting ligamentization and the health of grafts.

STUDY DESIGN: A hospital-based retrospective epidemiological study. OBJECTIVE: To examine the demographic and epidemiological characteristics of patients with TSCI in Guangdong to help health-related institutions develop measures to determine the best allocation of medical resources. SUMMARY OF BACKGROUND DATA: Traumatic spinal cord injury (TSCI) is a highly disabling and deadly injury. Currently, there is little information regarding the epidemiological characteristics for TSCI in Guangdong. METHODS: We retrospectively reviewed the medical records of partial second-grade class-A hospitals (mainly capturing general city and county hospitals and some large-scale affiliated hospitals) in Guangdong province according to the International Classification of Disease Version 10 (ICD-10) and diagnostic code of TSCI. RESULTS: The study included the medical records of 1,340 patients with TSCI, and the annual number of TSCI admissions increased during the 2003-2011 period. The male-to-female ratio was approximately 3.5:1. The major causes of spinal cord injuries were high falls (41.0%) and traffic accidents (37.8%). The most common injury among patients with TSCI was cervical injury (818 cases). In addition, 62.9% of the patients had spinal fractures, 24.0% had other fractures, and 13.7% had brain injuries. Furthermore, 25.1% (337/1,340) of the patients experienced clinical complications. The differences in the number of patients with and without complete injury who accepted surgery and hyperbaric oxygen therapy were statistically significant (P CONCLUSIONS: The epidemiological characteristics of TSCI in Guangdong has its own characteristics, and preventive measures should focus on high-risk populations, such as adult men. LEVEL OF EVIDENCE: 3. Language: en

Background The goals of this study were to explore the expression profiles and functional networks of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in mesenchymal stromal cells (MSCs) involved in regulating the function of monocytes and to clarify the mechanisms by which MSCs exert immunoregulatory effects on monocytes. Methods MSCs and CD14+ monocytes were separately isolated. The immunoregulatory effects of MSCs on monocytes were determined by flow cytometry. lncRNAs and mRNAs that were differentially expressed (DE) between the control group (MSCs only) and co-culture group (MSCs co-cultured with monocytes) were identified through high-throughput sequencing and bioinformatic analyses and were confirmed by qRT-PCR. Bioinformatic analyses were performed to identify the critical biological functions and signalling pathways involved in MSC-mediated monocyte regulation and to identify the functional networks formed between DE mRNAs and lncRNAs. Results MSCs showed a strong ability to induce monocyte migration but inhibited monocyte differentiation into M1 macrophages. A total of 145 DE lncRNAs and 768 DE mRNAs were identified between the control and co-culture groups. Significant fold changes in lncRNAs and mRNAs were confirmed by qRT-PCR. GO analysis demonstrated that DE mRNAs and lncRNAs were highly associated with terms related to binding and biological regulation. KEGG analysis revealed 122 significantly regulated pathways, including the cytokine-cytokine receptor pathway and chemokine signalling pathway. Interaction and co-expression networks were constructed for DE mRNAs and lncRNAs, and several key microRNAs were identified in the competitive endogenous RNA (ceRNA) network. Target genes of the DE lncRNAs were analysed to predict critical mRNA-lncRNA axes involved in the immunoregulatory function of MSCs. Conclusions Our research describes the lncRNA and mRNA expression profiles and functional networks involved in MSC-mediated regulation of monocytes. These results provide possible molecular mechanisms for the immunoregulatory function of MSCs and may help to elucidate possible molecular therapeutic targets in MSCs for the treatment of autoimmune diseases. Electronic supplementary material The online version of this article (10.1186/s13287-019-1306-x) contains supplementary material, which is available to authorized users.

The purpose of this study was to evaluate the effect of salmon calcitonin (sCT) on improving fibrosis-related indicators in frozen shoulder synovial/capsular fibroblasts (SCFs) and detect the potential downstream pathway. Quantitative real-time polymerase chain reaction and cell-substrate adhesion assays were used to measure alterations in fibrosis-related molecule expression and the cell adhesion ability of frozen shoulder SCFs after treatment with range concentrations of sCT. The presence of calcitonin receptors (CTRs) in shoulder joint synovial/capsular tissue samples was detected by immunohistochemistry (IHC). The downstream pathways of sCT in SCFs were further explored by utilizing three classical pathway inhibitors. With the addition of sCT to the culture medium of frozen shoulder SCFs, the messenger RNA (mRNA) expression of collagen type I (COL1A1), COL3A1, fibronectin 1, laminin 1, transforming growth factor-β1 (TGF-β1), and interleukin-1α (IL-1α) showed a descending trend as the sCT concentration increased. Treatment with sCT increased the expression of vascular endothelial growth factor and IL-6 in a dose-dependent manner. The enhanced adhesion ability of frozen shoulder SCFs gradually diminished with increasing concentrations of sCT. By using IHC, the CTR was detected extensively in the frozen shoulder joint synovium and capsule. Blocking the protein kinase C (PKC) pathway reversed the sCT-mediated suppression of COL1A1 production. Blocking the PKC or protein kinase A (PKA) pathway eliminated the sCT-induced inhibition of TGF-β1 production. This study demonstrated that sCT effectively improved the mRNA expression of fibrosis-related molecules and decreased the enhanced cell-substrate adhesion ability of frozen shoulder SCFs. sCT might achieve these effects by interacting with the CTR that is expressed on the SCF surface and by activating the downstream PKC or PKA pathway.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin (CaM)-dependent serine/threonine protein kinase, plays important roles in diverse apoptosis pathways not only in tumor suppression but also in neuronal cell death. The requirement of DAPK1 catalytic activity for its proposed cell functions and the elevation of catalytic activity of DAPK1 in injured neurons in models of neurological diseases, such as ischemia and epilepsy, validate that DAPK1 can be taken as a potential therapeutic target in these diseases. Recent studies show that DAPK1-NR2B, DAPK1-DANGER, DAPK1-p53, and DAPK1-Tau are currently known pathways in stroke-induced cell death, and blocking these cascades in an acute treatment effectively reduces neuronal loss. In this review, we focus on the role of DAPK1 in neuronal cell death after stroke. We hope to provide exhaustive summaries of relevant studies on DAPK1 signals involved in stroke damage. Therefore, disrupting DAPK1-relevant cell death pathway could be considered as a promising therapeutic approach in stroke.

Objective.We previously demonstrated that mesenchymal stem cells (MSC) from patients with ankylosing spondylitis (AS; ASMSC) have a greater osteogenic differentiation capacity than MSC from healthy donors (HDMSC) and that this difference underlies the pathogenesis of pathological osteogenesis in AS. Here we compared expression levels of long noncoding RNA (lncRNA) and mRNA between osteogenically differentiated ASMSC and HDMSC and explored the precise mechanism underlying abnormal osteogenic differentiation in ASMSC.Methods.HDMSC and ASMSC were induced with osteogenic differentiation medium for 10 days. Microarray analyses were then performed to identify lncRNA and mRNA differentially expressed between HDMSC and ASMSC, which were then subjected to bioinformatics analysis and confirmed by quantitative real-time PCR (qRT-PCR) assays. In addition, coding-non-coding gene co-expression (CNC) networks were constructed to examine the relationships between the lncRNA and mRNA expression patterns.Results.A total of 520 lncRNA and 665 mRNA were differentially expressed in osteogenically differentiated ASMSC compared with HDMSC. Bioinformatics analysis revealed 64 signaling pathways with significant differences, including transforming growth factor-β signaling. qRT-PCR assays confirmed the reliability of the microarray data. The CNC network indicated that 4 differentially expressed lncRNA, including lnc-ZNF354A-1, lnc-LIN54-1, lnc-FRG2C-3, and lnc-USP50-2 may be involved in the abnormal osteogenic differentiation of ASMSC.Conclusion.Our study characterized the differential lncRNA and mRNA expression profiles of osteogenically differentiated ASMSC and identified 4 lncRNA that may participate in the abnormal osteogenic differentiation of ASMSC. These results provide insight into the pathogenesis of pathological osteogenesis in AS.

Background Mesenchymal stem cells (MSCs) have been recognized as a promising tool for the treatment of various inflammatory disorders and autoimmune diseases. Stress conditions affect immune-mediated treatment and activate autophagy in MSCs. However, whether autophagy affects the MSC-mediated recruitment and differentiation of CD4+ T cells remains elusive. Methods MSCs were pretreated with 3-methyladenine (3-MA) and rapamycin to regulate autophagy, and then co-cultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of well-known chemokines were analyzed by quantitative real-time PCR. Enzyme-linked immunosorbent assays and western blot analysis were performed to detect C-X-C motif chemokine ligand 8 (CXCL8) and transforming growth factor (TGF)-β1 protein levels. An exogenous antibody and short hairpin RNA were used to regulate CXCL8 and TGF-β1 levels, which enabled us to evaluate how autophagy affected MSC-mediated CD4+ T cell migration and differentiation. Results 3-MA inhibited autophagy in MSCs, which was activated by rapamycin. Rapamycin increased the migration of CD4+ T cells, whereas 3-MA decreased their migration. Mechanistically, we found that autophagy strengthened CXCL8 secretion, and the addition of exogenous CXCL8 and an anti-CXCL8 antibody eliminated the difference of CD4+ T cell migration among groups. Further, the ratio of regulatory T (Treg) cells was increased in rapamycin-pretreated MSCs, but the ratio of T helper 1 (Th1) cells was decreased, while pretreatment of MSCs with 3-MA induced the opposite effect compared with the control group. TGF-β1 overexpression and knockdown using lentiviruses rectified the differences in the ratios of Treg and Th1 cells among the groups. Conclusion This study demonstrates that autophagy of mesenchymal stem cells mediates CD4+ T cell migration and differentiation through CXCL8 and TGF-β1, respectively. These results provide a potential new strategy for improving MSC-mediated therapy. Electronic supplementary material The online version of this article (10.1186/s13287-019-1380-0) contains supplementary material, which is available to authorized users.

Background: Rehabilitation is crucial for postoperative patients with low back pain (LBP). However, the implementation of traditional clinic-based programs is limited in developing countries, such as China, because of the maldistribution of medical resources. Mobile phone–based programs may be a potential substitute for those who have no access to traditional rehabilitation. Objective: The aim of this study was to examine the efficacy of mobile phone–based rehabilitation systems in patients who underwent lumbar spinal surgery. Methods: Patients who accepted spinal surgeries were recruited and randomized into 2 groups of rehabilitation treatments: (1) a mobile phone–based eHealth (electronic health) program (EH) or (2) usual care treatment (UC). The primary outcomes were (1) function and pain status assessed by the Oswestry Disability Index (ODI) and (2) the visual analog scale (VAS). Secondary outcomes were (1) general mental health and (2) quality of life (Likert scales, EuroQol-5 Dimension health questionnaire, and 36-item Short-Form Health Survey). All the patients were assessed preoperatively and then at 3, 6, 12, and 24 months postoperatively. Results: A total of 168 of the 863 eligible patients were included and randomized in this study. Our analysis showed that the improvement of primary outcomes in the EH group was superior to the UC group at 24 months postoperatively (ODI mean 7.02, SD 3.10, P

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases, inflammatory diseases, etc., due to their pleiotropic properties. However, largely incongruent data were obtained from different MSC-based clinical trials, which may be partially due to functional heterogeneity among MSC. Here, we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP), which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression, proliferation, migration, multipotency, immunomodulatory activity and global gene expression profile. Moreover, the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC), and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly, NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo, as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies.

Persistent neurotrophic factor delivery is crucial to create a microenvironment for cell survival and nerve regeneration in spinal cord injury (SCI). This study aimed to develop a NT-3/fibroin coated gelatin sponge scaffold (NF-GS) as a novel controlled artificial release therapy for SCI. In vitro, bone marrow-derived mesenchymal stem cells (MSCs) were planted into the NF-GS and release test showed that NF-GS was capable to generate a sustainable NT-3 release up to 28 days. MSCs in NF-GS had high cell activity with excellent cell distribution and phenotype. Then, the NF-GS was transplanted into the injury site of spinal cord of rat and canine in vivo, which exhibited strong biocompatibility during post-transplantation period. Four weeks following transplantation, the concentration of NT-3 was much higher than that in control groups. Cavity areas in the injury/graft site were significantly reduced due to tissue regeneration and axonal extensions associated with myelin sheath through the glial scar into the NF-GS. Additionally, the NF-GS decreased the inflammation by reducing the CD68 positive cells and TNF-α. A striking feature was the occurrence of some cells and myelin-like structure that appeared to traverse the NF-GS. The present results demonstrate that the NF-GS has the property to control the release of NT-3 from the NT-3/fibroin complex thus facilitating regeneration of injured spinal cord.

Low bone mineral density (BMD) is an important complication of ankylosing spondylitis (AS) that seriously affects men and their quality of life, even in young patients. However, the relationships among redox; levels of bone turnover markers (BTMs), inflammatory markers and disease activity; and low BMD in AS require clarification. We recruited 102 men aged 30-39 year with AS and 102 healthy, sex- and age-matched controls for this cross-sectional study. The subjects were analyzed for lumbar spine and femoral neck BMD by dual-energy X-ray absorptiometry. Significantly lower BMD and corresponding T-scores were observed in the AS patients compared with the controls (P < 0.05). The oxidant biomarker and antioxidant levels were significantly (P < 0.05) higher and lower, respectively, in the AS subjects compared with the controls, and the bone resorption and inflammatory marker levels were higher (P < 0.05). In subgroup analyses, the patients with osteoporosis or active disease had the highest levels of oxidant biomarkers (P < 0.05). Furthermore, the BMD T-scores in AS were found to be negatively correlated with oxidative status (P < 0.05). Multivariate binary logistic analysis showed that low BMD in the AS patients was associated with higher levels of advanced oxidation protein products, malondialdehyde and C-terminal telopeptide of type I collagen; lower levels of glutathione peroxidase; and higher scores of a bath ankylosing spondylitis metrology index. In conclusion, imbalanced redox was independently associated with low BMD in young men with AS and may play an important role in the pathogenesis of AS-related low BMD.

Emerging evidence has revealed that Nestin not only serves as a biomarker for multipotent stem cells, but also regulates cell proliferation and invasion in various tumors. However, the mechanistic contributions of Nestin to cancer pathogenesis are still unknown. In the present study, previously thought to reside exclusively in the cytoplasm, Nestin can also be found in the nucleus and participate in protecting tumor cells against cellular senescence. Specifically, we reveal that Nestin has a nuclear localization signal (aa318–aa347) at the downstream of rod domain. We then find nuclear Nestin could interact with lamin A/C. Mechanistic investigations demonstrate that Nestin depletion results in the activation of cyclin-dependent kinase 5 (Cdk5), which causes the phosphorylation of lamin A/C (mainly at S392 site) and its subsequent translocation to the cytoplasm for degradation. The findings establish a role for nuclear Nestin in tumor senescence, which involves its nucleus-localized form and interaction with lamin A/C., Nestin can be localised in the nucleus of cancer cells, but its nuclear role in tumorigenesis is unclear. Here, the authors show that nuclear Nestin prevents senescence in tumor cells by stabilising lamin A/C from proteasomal degradation to maintain nuclear integrity.

This study compared knee stability and alterations in the composition of synovial fluid among patients who underwent arthroscopic anterior cruciate ligament (ACL) reconstruction with either a hamstring autograft or a hamstring allograft. This prospective cohort study enrolled 175 patients. Of these patients, 90 underwent hamstring tendon autograft ACL reconstruction. The remaining patients (n=85) underwent hamstring tendon allograft ACL reconstruction. All of the patients had a minimum of 1 year of follow-up (mean, 2.5 years). Side-to-side differences and the proportion of mononuclear cells in knee joint synovial fluid were measured at 5 time points (preoperatively and at postoperative weeks 1, 3, 6, and 12). During the early postoperative phase, side-to-side knee laxity was greater among the allograft group compared with the autograft group ( P P >.05). These findings showed that, in the early postoperative phase, ACL reconstruction with a hamstring allograft resulted in greater knee laxity and immunologic response compared with the hamstring autograft group. However, at relatively long-term follow-up, both grafts achieved similar objective and subjective outcomes. [ Orthopedics. 2017; 40(5):e892–e897.]

Background context The understanding of vascular supply to the spinal cord is important given that the evolution of surgical approaches to the spine may bring along the potential for more frequent complications, especially a rare but devastating complication: that of spinal cord ischemia or infarction. To maximally avoid this complication, the relationship between the spinal cord vascularity and the anterior spine surgical approach needs further study. Purpose To provide a theoretical basis that will allow the spinal surgeon to take appropriate steps to avoid spinal cord ischemia during anterior spinal surgery through anatomic means. Study design Spinal cord vascular casting assessment with cadaveric specimen. Methods Twenty adult cadaveric specimens (11 men and 9 women) were obtained for the latex perfusion and vessel dissection. In addition, nine patients (seven men and two women) underwent superselective angiography of the spinal cord. The segmental arterial anastomosis and radiculomedullary vessels in the thoracolumbar region were shown and reviewed. Results There were approximately 21 pairs of segmental arteries in the thoracolumbar region. Adjacent segmental arteries were networked with each other. The latex infusion specimens demonstrated 72 anterior radiculomedullary arteries and 177 posterior radiculomedullary arteries in all 20 samples. The anterior and posterior spinal arteries were also networked with each other at several levels. Superselective spinal angiography was consistent with the latex infusion specimens showing. Conclusions The variety of anatomy of spinal cord arterial networks is shown, and the relation between the blood supply of certain spinal levels and the potential ischemic complications during the anterior surgical approach is discussed. It is hopefully of benefit to surgeons, after fully understanding the anatomy of these spinal vascular supply structures, that there may be even greater avoidance of vascular compromise in these challenging operations.

Nonhuman primate models of spinal cord injury (SCI) have been widely used in evaluation of the efficacy and safety of experimental restorative interventions before clinical trials. However, no objective methods are currently available for the evaluation of neural function in nonhuman primates. In our long-term clinical practice, we have used evoked potential (EP) for neural function surveillance during operation and accumulated extensive experience. In the present study, a nonhuman primate model of SCI was established in 6 adult cynomologus monkeys through spinal cord contusion injury at T8–T9. The neural function before SCI and within 6 months after SCI was evaluated based on EP recording. A scoring system including somatosensory evoked potentials (SSEPs) and transcranial electrical stimulation-motor evoked potentials (TES-MEPs) was established for the evaluation of neural function of nonhuman primates with SCI. We compared the motor function scores of nonhuman primates before and after SCI. Our results showed that the EP below the injury level significantly changed during the 6 months after SCI. In addition, a positive correlation was identified between the EP scores and motor function. The EP-based scoring system is a reliable approach for evaluating the motor function changes in nonhuman primates with SCI.

Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by pathological osteogenesis and inflammation. However, the pathogenesis of AS and the pathological relationship between osteogenesis and inflammation in this disease remain largely unknown. Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of osteogenic differentiation and immunoregulation. Recently, we demonstrated that MSCs from AS patients (ASMSCs) have a greater potential for osteogenic differentiation than MSCs from healthy donors (HDMSCs), which therefore seems to be a component of pathological osteogenesis in AS. Previous studies have indicated that the immunoregulatory abilities of MSCs change following differentiation. However, the subsequent effects of ASMSCs during abnormal osteogenic differentiation are unclear. Here, we further demonstrated that ASMSCs secreted more monocyte chemoattractant protein 1 (MCP1) than HDMSCs during osteogenic differentiation. This enhanced MCP1 secretion augmented monocyte migration, increased classical macrophage polarization, and enhanced TNF-α secretion. Inhibiting MCP1 secretion from osteogenic differentiated ASMSCs using lentiviruses encoding short hairpin RNAs ameliorated these dysfunctions. Blocking the ERK1/2 pathway in ASMSCs with U0126 corrected the abnormal osteogenic differentiation, inhibited MCP1 overexpression, and prevented subsequent monocyte dysfunction. Finally, MCP1 expression was up-regulated during osteogenic differentiation in ASMSCs in vivo and was locally augmented in osteoblasts at ossification sites in AS patients. In summary, our study determined that MCP1 overexpression during abnormal osteogenic differentiation of ASMSCs triggers monocyte dysfunctions. We propose the novel hypothesis that pathological osteogenesis can lead to inflammation in AS. This hypothesis may contribute to reveal the precise pathological relationship between osteogenesis and inflammation in the field of osteoimmunology.ASMSCs secreted more MCP1 during abnormal osteogenic differentiation. MCP1 overexpression leads to monocyte dysfunctions. Pathological osteogenesis can lead to inflammation in AS.

This is a retrospective analysis of the strategy and clinical results of surgery combined with intraoperative radiotherapy (IORT) to treat spinal metastases. We delivered tumour-conformal IORT in 40 patients with 52 metastatic vertebrae based on our surgical classification system. The strategies were evaluated with respect to neurologic function and spinal stability. The EORTC QLQ-BM22, visual analogue scale (VAS) and the Frankel Scale were used to assess quality of life, pain and neurologic function. Local control was evaluated every 3 months using X-rays and MRI. Micro-invasive IORT was performed in 42 vertebrae (80.8%), and open surgery with IORT was performed in 10 vertebrae (19.2%). Single-level, 2-level and 3-level IORT was performed in 30, 8 and 2 cases, respectively. The delivered dose was 9.2 ± 3.6 Gy (8–15 Gy) with a depth of 10.1 ± 2.1 mm. The actual IORT treatment time was 5 min and 16 s. The follow-up period was 6–23 months (mean: 12.5 months). The local control rate was 92.3%. The EORTC QLQ-BM22 scores showed that patients had significant improvements in pain location, degree and function after treatment (P

Background Arthroscopic anterior cruciate ligament (ACL) reconstruction with allograft may vary in instrumented laxity compared with autograft. T helper (Th) cells (CCR4+CCR6+Th) and regulatory T (Treg) cells are important in the early stage of immunologic reactions. It is unknown whether the ratio of CCR4+CCR6+Th to Treg is imbalanced and if it correlates with early postoperative laxity after ACL allograft reconstruction. We investigated the ratio and functional influence between these cells and the correlation with postoperative anterior knee laxity. Methods In 40 patients who experienced unilateral arthroscopy-assisted ACL reconstruction, the ACL graft was an allograft in 20 patients and autograft in 20 patients. Maximum manual anterior tibiofemoral laxity measurements were performed with arthrometry testing. The phenotypes of CCR4+CCR6+Th and Treg cells of peripheral blood and synovial fluid from the two patient groups were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in (3)H-thymidine proliferation assays. Results Both CCR4+CCR6+Th and Treg cells were significantly increased in the synovial fluid in the allograft group and were correlated with anterior knee laxity. The ratio of CCR4+CCR6+Th to Treg cells in the synovial fluid was positively correlated with laxity. Synovial CD4+CD25+ Treg cells displayed an increased suppressive capacity compared with blood CD4+CD25+ Treg cells. Activated responder T cells from the synovial fluid were less susceptible to CD4+CD25+ T cell-mediated suppression than were responder cells from blood. Conclusions The ratio of CCR4+CCR6+Th to Treg cells in the synovial fluid was correlated with anterior laxity after ACL allograft reconstruction.

Osteosarcoma is the most frequent primary malignant tumor of the skeleton and occurs mainly in children and adolescent. The prognosis of osteosarcoma is very poor due to its aggressive and no effective treatment. This study is the first to investigate the anti-cancer effects of antisense pEGFP-C1-Survivin on human osteosarcoma cells. It was shown in our results that Survivin blockaded could significantly induce apoptosis and inhibit the invasive of osteosarcoma cells line MG-63. The effects were probably produced by the decreased expression of Survivin induced by antisense pEGFP-C1-Survivin which was examined by RT-PCR and western blotting. All these suggested that Survivin should be very important in the development of osteosarcoma and Survivin blockaded by using antisense pEGFP-C1-Survivin could markedly inhibit the proliferation and invasion of osteosarcoma cells line MG-63, partially reversed their malignant phenotype. Targeting Survivin might be a promising option in the treatment of osteosarcoma.

Bone marrow-derived mesenchymal stem cells (BMSCs) are being broadly investigated for treating numerous inflammatory diseases. However, the low survival rate of BMSCs during the transplantation process has limited their application. Autophagy can maintain cellular homeostasis and protect cells against environmental stresses. Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine that can induce both autophagy and apoptosis of BMSCs. However, the actual role of autophagy in TNF-α-induced apoptosis of BMSCs remains poorly understood. In the current study, BMSCs were treated with TNF-α/cycloheximide (CHX), and cell death was examined by the Cell Counting Kit-8, Hoechst 33342 staining, and flow cytometric analysis as well as by the level of caspase-3 and caspase-8. Meanwhile, autophagic flux was examined by analyzing the level of microtubule-associated protein light chain 3 B (LC3B)-II and SQSTEM1/p62 and by examining the amount of green fluorescent protein-LC3B by fluorescence microscopy. Then, the cell death and autophagic flux of BMSCs were examined after pretreatment and cotreatment with 3-methyladenine (3-MA, autophagy inhibitor) or rapamycin (Rap, autophagy activator) together with TNF-α/CHX. Moreover, BMSCs pretreated with lentiviruses encoding short hairpin RNA of beclin-1 (BECN1) were treated with TNF-α/CHX, and then cell death and autophagic flux were detected. We showed that BMSCs treated with TNF-α/CHX presented dramatically elevated autophagic flux and cell death. Furthermore, we showed that 3-MA and shBECN1 treatment accelerated TNF-α/CHX-induced apoptosis, but that Rap treatment ameliorated cell death. Our results demonstrate that autophagy protects BMSCs against TNF-α-induced apoptosis. Enhancing the autophagy of BMSCs may elevate cellular survival in an inflammatory microenvironment.

To evaluate the clinical outcomes of total en-bloc spondylectomy (TES) in recurrence spinal tumor.The study was a retrospective study of recurrence spinal tumor from January 2010 to October 2013. A total of 6 patients with recurrent spinal tumor underwent TES procedures, with 5 cases located in thoracic spine and 1 case located in L1. There were 3 male and 3 female patients, with a mean age of 33.2 years. Pathological diagnosis included giant cell tumor of bone in 3 cases, breast cancer, lung cancer and nasopharyngeal carcinoma with 1 case in each. The operation time, bleeding loss, resected segments, cutting edge, spinal cord function and complications was evaluated.Single segment resected in 1 case, 2 segments resected in 2 cases and 3 segments resected in 3 cases. The average operation time was 8.9 hours (7.5 to 12.0 hours). The average blood loss was 3 116 ml (2 500 to 4 500 ml). The average follow-up period was 23.2 months (12 to 47 months) without recurrence. There was no spinal cord injury during operation. The neurologic function was significantly improved in 2 cases (American Spinal Injury Association (ASIA) grade C to grade D), unchanged in 1 cases (ASIA grade B) and no deteriorated case in 3 cases (ASIA grade E). There was no perioperative deaths case. Complications included 2 cases pleural rupture, 1 case dural tear and 1 case massive haemothorax. No peri-operation death case.Some of the recurrent spinal tumors are still suitable for en-bloc resection and TES procedure with the extent of its applicability under strict control.

To study the osteogenic differentiation capacity of bone marrow-derived mesenchymal stem cells (BM-MSCs) from patients with ankylosing spondylitis (AS) and to investigate the mechanisms of abnormal osteogenic differentiation of BM-MSCs in AS.BM-MSCs from healthy donors (HD-MSCs) and patients with AS (AS-MSCs) were cultured in osteogenic differentiation medium for 0-21 days, after which their osteogenic differentiation capacity was determined using alizarin red S and alkaline phosphatase assays. Gene expression levels of osteoblastic markers and related cytokines were detected by high-throughput quantitative reverse transcription-polymerase chain reaction. Enzyme-linked immunosorbent assay was performed to detect protein levels of bone morphogenetic protein 2 (BMP-2) and Noggin in the cell culture supernatant. The activation of Smad1/5/8 and MAPK signaling pathways was measured by Western blotting. The balance between BMP-2 and Noggin expression was regulated using lentiviruses encoding short hairpin RNA and exogenous Noggin, respectively, which enabled evaluation of how this balance affected osteogenic differentiation of AS-MSCs.AS-MSCs outperformed HD-MSCs in osteogenic differentiation capacity. During osteogenic differentiation, AS-MSCs secreted more BMP-2 but less Noggin, accompanied by an overactivation of Smad1/5/8 and ERK-1/2. When the Noggin concentration was increased or BMP-2 expression was inhibited, the abnormal osteogenic differentiation of AS-MSCs was rectified. In addition, the balance between BMP-2 and Noggin secretion was restored.The results of this study demonstrate that an imbalance between BMP-2 and Noggin secretion induces abnormal osteogenic differentiation of AS-MSCs. These findings reveal a mechanism of pathologic osteogenesis in AS and provide a new perspective on inhibiting pathologic osteogenesis by regulating the balance between BMP-2 and Noggin.