Exosomal long non-coding RNA TRAFD1-4:1 derived from fibroblast-like synoviocytes suppresses chondrocyte proliferation and migration by degrading cartilage extracellular matrix in rheumatoid arthritis.

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

Endoplasmic reticulum stress-dependent ROS production mediates synovial myofibroblastic differentiation in the immobilization-induced rat knee joint contracture model.

Joint contracture is a common complication for people with joint immobility that involves fibrosis structural alteration in the joint capsule. Considering that endoplasmic reticulum (ER) stress plays a prominent role in the promotion of tissue fibrosis, we investigated whether the unfolded protein response (UPR) contributes to the fibrotic development in immobilization-induced knee joint contractures. Using a non-traumatic rat knee joint contracture model, twelve female Sprague-Dawley rats received knee joint immobilization for a period of 8 weeks. We found that fibrosis protein markers (type I collagen, α-SMA) and UPR (GRP78, ATF6α, XBP1s) markers were parallelly upregulated in rat primary cultured synovial myofibroblasts. In the same cell types, pre-treatment with an ER stress inhibitor, 4-phenylbutyric acid (4-PBA), not only abrogated cytokine TGFß1 stimulation but also reduced the protein level of UPR. Additionally, high reactive oxygen species (ROS) generation was detected in synovial myofibroblasts through flow cytometry, as expected. Notably, TGFß1-induced UPR was significantly reduced through the inhibition of ROS with antioxidants. These data suggest that ER stress act as a pro-fibrotic stimulus through the overexpression of ROS in synovial fibroblasts. Interestingly, immunohistochemical results showed an increase in the UPR protein levels both in human acquired joint contractures capsule tissue and in animal knee joint contracture tissue. Together, our findings suggest that ER stress contributes to synovial myofibroblastic differentiation in joint capsule fibrosis and may also serve as a potential therapeutic target in joint contractures.

HGF/c-Met signaling mediated mesenchymal stem cell-induced liver recovery in intestinal ischemia reperfusion model.

PURPOSE: Liver injury triggered by intestinal ischemia-reperfusion (IIR) usually presage multiorgan dysfunction and death in patients. Recent studies suggest mesenchymal stem cells (MSCs) possess a protective potential against organ damage. Since relative evidence is insufficient and the mechanism is not well understood, we investigated the effect of hepatocyte growth factor c-Met signaling (HGF/c-Met) on recruitment of MSCs and subsequent protection against liver injury triggered by IIR in a rat model. METHODS: IIR models were built as rats were subjected to 75 min of superior mesenteric artery occlusion and subsequent 4 h reperfusion. Either of pure MSCs and MSCs pretreated with HGF or SU11274 (c-Met inhibitor) were injected into rat models. Biochemical and pathologic alterations of liver in IIR model were measured to evaluate the therapeutic effect of MSCs and drug treatment. Concurrently, the effect of HGF and SU11274 on c-Met and phosphorylated Met expression in MSCs and MSCs migration were assessed in in vitro experiment. RESULTS: IIR-induced liver injury was manifested by significant increase in serum ALT, AST and HGF levels as well as pathological change. MSCs with highly c-Met expression ameliorated the increase of serum transaminase levels and hepatic histopathological change, while SU11274 weaken these effects. HGF upregulated c-Met and phosphorylated Met expression in MSCs and enhanced its liver protection effect. Transwell assays demonstrated HGF promoted MSCs migration, which was blocked by SU11274. CONCLUSIONS: HGF/c-Met signaling pathway plays an essential role in the homing of MSCs towards injured liver triggered by intestinal ischemia-reperfusion, and then mediates MSC-induced liver repair.

Study on Shoulder Joint Parameters and Available Supraspinatus Outlet Area Using Three-Dimensional Computed Tomography Reconstruction.

Studies addressing the anatomical values of the supraspinatus outlet area (SOA) and the available supraspinatus outlet area (ASOA) are insufficient. This study focused on precisely measuring the SOA and ASOA values in a sample from the Chinese population using 3D CT (computed tomography) reconstruction. We analyzed CT imaging of 96 normal patients (59 males and 37 females) who underwent shoulder examinations in a hospital between 2011 and 2021. The SOA, ASOA, acromiohumeral distance (AHD), coracohumeral distance (CHD), coracoacromial arch radius (CAR), and humeral head radius (HHR) were estimated, and statistical correlation analyses were performed. There were significant sex differences observed in SOA (men: 957.62 ± 158.66 mm2; women: 735.87 ± 95.86 mm2) and ASOA (men: 661.35 ± 104.88 mm2; women: 511.49 ± 69.26 mm2), CHD (men: 11.22 ± 2.24 mm; women: 9.23 ± 1.35 mm), CAR (men: 37.18 ± 2.70 mm; women: 33.04 ± 3.15 mm), and HHR (men: 22.65 ± 1.44 mm; women: 20.53 ± 0.95 mm). Additionally, both SOA and ASOA showed positive and linear correlations with AHD, CHD, CAR, and HHR (R: 0.304-0.494, all p < 0.05). This study provides physiologic reference values of SOA and ASOA in the Chinese population, highlighting the sex differences and the correlations with shoulder anatomical parameters.

Intervertebral disk degeneration: T1ρ MR imaging of human and animal models.

PURPOSE: To evaluate disk degeneration in human and animal models by using a T1ρ magnetic resonance (MR) imaging technique to help in understanding the natural history and progression of intervertebral disk degeneration. MATERIALS AND METHODS: After institutional review board approval was obtained, 80 subjects (54 men and 26 women; mean age ± standard deviation, 31.6 years ± 6.20) with 400 lumbar intervertebral disks were examined at MR imaging. With approval from the animal care committee, six rhesus monkeys received two levels of either annulus fibrosus puncture or pingyangmycin subendplate injection at L3-4 and L5-6 to mimic disk degeneration. Lumbar spines of all the animals were examined at radiography and MR imaging preoperatively and 1 day and 1, 3, 6, 9, and 13 months postoperatively. Pfirrmann grading system and T1ρ quantification were used to evaluate the degenerative degree of the disks of both humans and animals. RESULTS: The mean T1ρ values of lumbar intervertebral disks of human subjects were 136.0 msec ± 31.4 and 76.1 msec ± 14.2 at Pfirrmann grades II and III, respectively. The T1ρ values in lumbar intervertebral disks of the rhesus monkey models of disk degeneration had a rapid decrease from approximately 110 msec to 80 msec and then tended to stabilize after operation. There was a large T1ρ value decrease between Pfirrmann grades II and III in human subjects that coincided with the rapid degeneration process of lumbar intervertebral disks in the rhesus monkeys. Pfirrmann grades were significantly correlated with T1ρ values in both humans (r = -0.681, P < .001) and rhesus monkeys (r = -0.824, P < .001). CONCLUSION: The data demonstrate that rapid intervertebral disk degeneration occurs early in the degenerative cascade, between Pfirrmann grades II and III.

Is surgery for spine metastasis reasonable in patients older than 60 years?

BACKGROUND: Spinal metastases are common in patients older than 60 years with cancer. Because of the uncertainty of survival and the high incidence of fatal complications, however, chemotherapy and radiotherapy generally have been considered preferable and surgery a treatment of last resort for these patients. Further, the selection criteria indicating surgery and reliable prognostic factors for survival remain controversial. QUESTIONS/PURPOSES: We therefore assessed surgical complications, postoperative function, and risk factors affecting their overall survival. METHODS: We retrospectively reviewed 92 patients 60 years or older (range, 60-81 years) who had surgery for spinal metastases. The surgical complications were recorded and a VAS pain score, Frankel grade, and Karnofsky score were obtained. Statistical analyses were performed to identify factors associated with survival. The minimum followup was 6 months (mean, 22 months; range, 6-78 months). RESULTS: Surgical complications occurred in 21 patients. Pain levels decreased postoperatively in 90% of patients and neurologic function improved in 78%. The Karnofsky status improved in 58 patients giving an improvement rate of 63%. The overall survival rates at 1 year and 3 years were 61% and 35% with a median of 15 months. Primary tumor type and Tokuhashi score independently predicted survival in patients with spinal metastases. CONCLUSION: Our findings suggest surgery for spinal metastasis can achieve pain relief, neurologic improvement, and restoration of general condition but with a high risk of complications. Primary tumor type and Tokuhashi scoring independently predicted survival in patients with spinal metastases after surgery. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Injectable Decorin/Gellan Gum Hydrogel Encapsulating Adipose-Derived Stem Cells Enhances Anti-Inflammatory Effect in Cartilage Injury via Autophagy Signaling.

Adipose-derived stem cells (ADSCs) are employed as a promising alternative in treating cartilage injury. Regulating the inflammatory "fingerprint" of ADSCs to improve their anti-inflammatory properties could enhance therapy efficiency. Herein, a novel injectable decorin/gellan gum hydrogel combined with ADSCs encapsulation for arthritis cartilage treatment is proposed. Decorin/gellan gum hydrogel was prepared according to the previous manufacturing protocol. The liquid-solid form transition of gellan gum hydrogel is perfectly suitable for intra-articular injection. Decorin-enriched matrix showing an immunomodulatory ability to enhance ADSCs anti-inflammatory phenotype under inflammation microenvironment by regulating autophagy signaling. This decorin/gellan gum/ADSCs hydrogel efficiently reverses interleukin-1ß-induced cellular injury in chondrocytes. Through a mono-iodoacetate-induced arthritis mice model, the synergistic therapeutic effect of this ADSCs-loaded hydrogel, including inflammation attenuation and cartilage protection, is demonstrated. These results make the decorin/gellan gum hydrogel laden with ADSCs an ideal candidate for treating inflammatory joint disorders.

The footprint of the anterior horn of medial meniscus: A novel and reliable landmark for the tibial vertical cut in Oxford mobile-bearing medial unicompartmental knee arthroplasty.

BACKGROUND: Tibial vertical cut is crucial for rotational position and bony coverage in Oxford mobile-bearing medial unicompartmental knee arthroplasty (UKA). This study aimed to determine whether the footprint of the anterior horn of medial meniscus (FAM) is a reliable landmark for tibial vertical cut. METHODS: The FAM and the line through FAM and the edge of anterior cruciate ligament insertion (FAMA line) were identified by dissection five knee joint specimens. The angle between FAMA line and standard Akagi's line was measured. From 2022 to 2023, 64 patients (74 knees) diagnosed as anteromedial osteoarthritis were included to undergo primary Oxford medial UKA by two surgeons (Group 1 and 2), using FAMA line as a landmark for tibial vertical cut. The anteroposterior (AP) length, mediolateral (ML) length of tibial cut and tibial prothesis were measured by vernier caliper. ML/AP ratio was also calculated, and data were compared intragroup and intergroup. Mediolateral position and external rotation of tibial components were assessed postoperatively. RESULTS: FAMA line was parallel to standard Akagi's line. No significant differences were found in AP and ML lengths between tibial cut and tibial component (AP different value = 0.007 ± 0.154 cm, P = 0.674, ML different value = 0.020 ± 0.195 cm, P = 0.155). The ML/AP ratio was similar between the two groups (P = 0.141, 0.646, 0.255, 0.607, 0.384, size AA âˆ¼ D). No significant difference was found in mediolateral position (0.87 ± 0.03 vs. 0.86 ± 0.03, P = 0.156) and external rotation (6.88 ± 2.08 vs. 6.68 ± 2.22, P = 0.746) of the tibial component between the two groups. CONCLUSION: The FAM is a reliable landmark for tibial vertical cut in Oxford UKA.

Poly-l-lactide-co-ε-caprolactone (PLCL) and poly-l-lactic acid (PLLA)/gelatin electrospun subacromial spacer improves extracellular matrix (ECM) deposition for the potential treatment of irreparable rotator cuff tears.

Biodegradable subacromial spacer implantation has become practicable for the treatment of irreparable rotator cuff tears (IRCT). However, the relative high degradation rate and inferior tissue regeneration properties of current subacromial spacer may lead to failure regards to long-term survival. It is reported that satisfactory clinical results lie in the surrounding extracellular matrix (ECM) deposition after implantation. This study aims to develop a biological subacromial spacer that would enhance tissue regeneration properties and results in better ECM deposition. Physicochemical properties were characterized on both poly-l-lactide-co-ε-caprolactone (PLCL) dip-coating spacer (monolayer spacer, MS) and PLCL dip-coating + Poly-l-Lactic Acid (PLLA)/Gelatin electrospun spacer (Bilayer Spacer, BS). Cytocompatibility, angiogenesis, and collagen inducibility were evaluated with tendon fibroblasts and endothelial cells. Ultrasonography and histomorphology were used to analyze biodegradability and surrounding ECM deposition after the implantation in vivo. BS was successfully fabricated with the dip-coating and electrospinning technique, based on the human humeral head data. In vitro studies demonstrated that BS showed a greater cytocompatibility, and increased secretion of ECM proteins comparing to MS. In vivo studies indicated that BS promoted ECM deposition and angiogenesis in the surrounding tissue. Our research highlights that BS exhibits better ECM deposition and reveals a potential candidate for the treatment of IRCT in future.

In Vivo Anatomical Research by 3D CT Reconstruction Determines Minimum Acromiohumeral, Coracohumeral, and Glenohumeral Distances in the Human Shoulder: Evaluation of Age and Sex Association in a Sample of the Chinese Population.

Accurate measurement of the minimum distance between bony structures of the humeral head and the acromion or coracoid helps advance a better understanding of the shoulder anatomical features. Our goal was to precisely determine the minimum acromiohumeral distance (AHD), coracohumeral distance (CHD), and glenohumeral distance (GHD) in a sample of the Chinese population as an in vivo anatomical analysis. We retrospectively included 146 patients who underwent supine computed tomography (CT) examination of the shoulder joint. The minimum AHD, CHD, and GHD values were quantitatively measured using three-dimensional (3D) CT reconstruction techniques. The correlation between minimum AHD, CHD, and GHD value and age with different sexes was evaluated using Pearson Correlation Coefficient. The mean value of minimum AHD in males was greater than that in females (male 7.62 ± 0.98 mm versus female 7.27 ± 0.86 mm, p = 0.046). The CHD among different sexes differed significantly (male 10.75 ± 2.40 mm versus female 8.76 ± 1.38 mm, p < 0.001). However, we found no statistical differences in GHD with different sexes (male 2.00 ± 0.31 mm versus female 1.96 ± 0.36 mm, p > 0.05). In terms of age correlation, a negative curve correlation existed between age and AHD among the different sexes (male R2 = 0.124, p = 0.030, female R2 = 0.112, p = 0.005). A negative linear correlation was found in CHD among the different sexes (male R2 = 0.164, p < 0.001, female R2 = 0.122, p = 0.005). There were no differences between age and minimum GHD in both sexes. The 3D CT reconstruction model can accurately measure the minimum AHD, CHD, and GHD value in vivo and is worthy of further investigation for standard clinical anatomical assessment. Aging may correlate with AHD and CHD narrowing for both sexes.

The Regenerative Role of Gelatin in PLLA Electrospun Membranes for the Treatment of Chronic Massive Rotator Cuff Injuries.

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

Exosomal HMGB1 derived from hypoxia-conditioned bone marrow mesenchymal stem cells increases angiogenesis via the JNK/HIF-1α pathway.

Mesenchymal stem cells (MSCs) have been described to induce angiogenesis in various tissues and have been used for the development of novel cell-based therapies. Increasing evidence suggests that MSCs execute their paracrine function via the secretion of exosomes, especially under hypoxic conditions. However, the mechanisms by which MSC-derived exosomes secreted under hypoxia enhance angiogenesis still remain unclear. To study exosome physiology under hypoxic or normoxic conditions, we isolated exosomes from bone marrow mesenchymal stem cells (BMSCs). Furthermore, we detected the uptake of exosomes by human umbilical vein endothelial cells (HUVECs) by immunofluorescence staining. In addition, we determined the effects of exosomes on cell viability, migration and tube formation in HUVECs by Cell Counting Kit-8, migration and tube formation assays, respectively. We examined the expression of key proteins related to exosome-induced angiogenesis by BMSCs cultured under hypoxic conditions by western blot. Exosomes released by BMSCs cultured under hypoxic conditions enhanced cell proliferation, migration and angiogenesis of HUVECs. Hypoxia induced the expression of high mobility group box 1 protein (HMGB1) in BMSC-derived exosomes, and silencing of HMGB1 abolished the angiogenic effect in HUVECs. Furthermore, exosomal HMGB1 activated the JNK signaling pathway and induced hypoxia-inducible factor-1α/vascular endothelial growth factor expression, consequently enhancing angiogenesis in HUVECs. Our data reveal that exosomal HMGB1 promotes angiogenesis via JNK/hypoxia-inducible factor-1α signaling. Therefore, BMSC exosomes derived under hypoxia may have potential for development of novel treatment strategies for angiogenesis-related diseases.

3D bioprinting of integral ADSCs-NO hydrogel scaffolds to promote severe burn wound healing.

Severe burns are challenging to heal and result in significant death throughout the world. Adipose-derived mesenchymal stem cells (ADSCs) have emerged as a promising treatment for full-thickness burn healing but are impeded by their low viability and efficiency after grafting in vivo. Nitric oxide (NO) is beneficial in promoting stem cell bioactivity, but whether it can function effectively in vivo is still largely unknown. In this study, we bioprinted an efficient biological scaffold loaded with ADSCs and NO (3D-ADSCs/NO) to evaluate its biological efficacy in promoting severe burn wound healing. The integral 3D-ADSCs/NO hydrogel scaffolds were constructed via 3D bioprinting. Our results shown that 3D-ADSCs/NO can enhance the migration and angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs). Burn wound healing experiments in mice revealed that 3D-ADSCs/NO accelerated the wound healing by promoting faster epithelialization and collagen deposition. Notably, immunohistochemistry of CD31 suggested an increase in neovascularization, supported by the upregulation of vascular endothelial growth factor (VEGF) mRNA in ADSCs in the 3D biosystem. These findings indicated that 3D-ADSC/NO hydrogel scaffold can promote severe burn wound healing through increased neovascularization via the VEGF signalling pathway. This scaffold may be considered a promising strategy for healing severe burns.

[Predictive value of three surgical scoring systems for estimation of life expectancy in patients with extradural spinal metastasis].

OBJECTIVE: To evaluate the predictive values of Tokuhashi score, revised Tokuhashi score and Tomita score systems for life expectancy and treatment options in patients with spinal metastasis. METHODS: From February 1996 to January 2009, spinal operations in 104 cases with spinal metastasis were performed in our hospital. There were 65 males and 39 females, with an average of 53.4 years (median 52.5 years). To calculate AUC (area under the curve) values of Receiver Operating Characteristic (ROC) curves of three scores, and to analyze the accuracy of prediction of life expectancy. To compare the actual survival time with the expected survival time of the three scores by Kaplan-Meier method. Spearman correlation analysis was performed between the survival time and three scoring systems. RESULTS: All cases were followed-up with an average duration of 10.9 months, and 77 patients died. AUC analysis of ROC curves showed that the difference of the accuracy of the three scores was not significant. AUC in all groups of Tokuhashi Score was low, with a poor diagnostic accuracy. In the "died within 3 months" and "died within 6 months" groups of revised Tokuhashi score, the accuracy was low, while high in the other two groups. The AUC values of Tomita score in "died within 6 months" and "died within 24 months" were high, with a great diagnostic accuracy while the other two groups were low with a low diagnostic accuracy. Kaplan-Meier survival curve analysis showed that the actual survival time in all three scores was not entirely consistent with the expected survival time. Tokuhashi score and revised score were positively correlated with the survival time while that of Tomita score was negative. CONCLUSION: All the three prognosis scores in patients with spinal metastasis were closely related with survival time. The combination of Tokuhashi score and Tomita score may be applied to better predict postoperative survival prognosis and guide the surgical options for patients with spinal metastasis.

Lumican promotes joint fibrosis through TGF-ß signaling.

Joint contracture (also known as arthrofibrosis) is a fibrotic joint disorder characterized by excessive collagen production to form fibrotic scar tissue and adhesions within joint capsules. This can severely affect day-to-day activities and quality of life because of a restricted range of motion in affected joints. The precise pathogenic mechanism underlying joint contractures is not fully understood. Lumican belongs to the class II small leucine-rich repeat proteoglycan superfamily, which makes up collagen fibrils in the extracellular matrix. Lumican is ubiquitously expressed in the skin, liver, heart, uterus and articular cartilage and has reported roles in cell migration, proliferation, angiogenesis and Toll-like receptor 4 signaling. Previous research has suggested that lumican is involved in the pathogenesis of several fibrotic diseases. Because joint contracture resembles a fibrotic disease, we aimed to investigate the role of lumican in the development of joint contracture in vitro. Here, we showed that protein levels were up-regulated in the fibrotic joint capsule versus control. We observed that lumican significantly enhanced the proliferation, migration and fibroblast-myofibroblast transition of synovial fibroblasts. Moreover, lumican led to increased transcription of alpha-smooth muscle actin, matrix metallopeptidase 9, Collagen I, plasminogen activator inhibitor 1 and transforming growth factor-ß in vitro. Lumican treatment promoted collagen lattice contraction in a dose-dependent manner as early as 24 h after treatment. Thus, our studies reveal that lumican could promote fibroblast-myofibroblast transition and joint contracture.

Exosomes from 3D culture of marrow stem cells enhances endothelial cell proliferation, migration, and angiogenesis via activation of the HMGB1/AKT pathway.

BACKGROUND: Angiogenesis is an essential step in tissue engineering. MSC exosomes play an important role in angiogenesis. Functional biomolecules in exosomes vested by the culture microenvironment can be transferred to recipient cells and affects their effect. 3D culture can improve the proliferation and activity of MSCs. However, whether exosomes derived from 3D culture of MSCs have an enhanced effect on angiogenesis is unclear. METHODS: Herein, we compared the bioactivity of exosomes produced by conventional 2D culture (2D-exos) and 3D culture (3D-exos) of bone marrow stem cells (BMSCs) in angiogenesis. RESULTS: A series of in vitro and in vivo experiments indicated that 3D-exos exhibited stronger effects on HUVEC cell proliferation, migration, tube formation, and in vivo angiogenesis compared with 2D-exos. Moreover, the superiority of 3D-exos might be attributed to the activation of HMGB1/AKT signaling. CONCLUSIONS: These results indicate that exosomes from 3D culture of MSCs may serve as a potential therapeutic approach for pro-angiogenesis.

Comprehensive Analysis of a ceRNA Network Identifies lncR-C3orf35 Associated with Poor Prognosis in Osteosarcoma.

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

Anti-osteosarcoma property of decorin-modified titanium surface: A novel strategy to inhibit oncogenic potential of osteosarcoma cells.

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

TIMP-1 inhibits proliferation and osteogenic differentiation of hBMSCs through Wnt/ß-catenin signaling.

The present study aimed to evaluate the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1) on the proliferation and osteogenic differentiation potential of human bone marrow-derived MSCs (hBMSCs). hBMSCs with stable TIMP-1 overexpression or TIMP-1 knockdown were generated. Osteogenic differentiation was assessed by Alizarin Red S staining, alkaline phosphatase (ALP) activity and expression of specific markers. Compared with the vehicle controls, TIMP-1 knockdown significantly promoted the growth of hBMSCs. TIMP-1 knockdown up-regulated ß-catenin and cyclin D1 proteins. During osteogenic differentiation, TIMP-1 knockdown elevated the deposition of calcium nodules, ALP activity and the mRNA levels of the osteogenic markers sex determining region Y-box 9 (Sox9), CCAAT-enhancer-binding protein and peroxisome proliferator-activated receptor γ. During osteogenic differentiation, TIMP-1 knockdown significantly enhanced the up-regulation of osteocalcin proteins. Meanwhile, TIMP-1 overexpression attenuated the Wnt/activator Wnt3a-induced up-regulation cyclin D1 and Runt-related transcription factor 2 (RUNX-2) (during osteogenic differentiation) proteins, while TIMP-1 knockdown restored the inhibitor Dickkopf 1-induced inhibition effect on the expression of ß-catenin, cyclin D1 and RUNX-2. TIMP-1 plays a negative regulatory role in the proliferation and osteogenesis of hBMSCs, at least partially, through Wnt/ß-catenin signaling.

Autograft microskin combined with adipose-derived stem cell enhances wound healing in a full-thickness skin defect mouse model.

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