[Retracted] MicroRNA­124 acts as a tumor­suppressive miRNA by inhibiting the expression of Snail2 in osteosarcoma.

[This retracts the article DOI: 10.3892/ol.2018.7994.].

Extracellular vesicles in tumorigenesis, metastasis, chemotherapy resistance and intercellular communication in osteosarcoma.

HIGHLIGHTS: Extracellular vehicles play crucial function in osteosarcoma tumorigenesis.Extracellular vehicles mediated the intercellular communication of osteosarcoma cells with other types cells in tumor microenvironment.Extracellular vehicles have potential utility in osteosarcoma diagnosis and treatment.

Modification of mesenchymal stem cells for cartilage-targeted therapy.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by the destruction of the articular cartilage, sclerosis of the subchondral bone, and joint dysfunction. Its pathogenesis is attributed to direct damage and mechanical destruction of joint tissues. Mesenchymal stem cells (MSCs), suggested as a potential strategy for the treatment of OA, have shown therapeutic effects on OA. However, the specific fate of MSCs after intraarticular injection, including cell attachment, proliferation, differentiation, and death, is still unclear, and there is no guarantee that stem cells can be retained in the cartilage tissue to enact repair. Direct homing of MSCs is an important determinant of the efficacy of MSC-based cartilage repair. Recent studies have revealed that the unique homing capacity of MSCs and targeted modification can improve their ability to promote tissue regeneration. Here, we comprehensively review the homing effect of stem cells in joints and highlight progress toward the targeted modification of MSCs. In the future, developments of this targeting system that accelerate tissue regeneration will benefit targeted tissue repair.

A minor review of microRNA-338 exploring the insights of its function in tumorigenesis.

MicroRNAs(miRNAs) are small non-coding RNAs which have a critical role in various biological processes via direct binding and post-transcriptionally regulating targeted genes expression. More than one-half of human genes were regulated by miRNAs and their aberrant expression was detected in various human diseases, including cancers. miRNA-338 is a new identified miRNA and increasing evidence show that miRNA-338 participates in the progression of lots of cancers, such as lung cancer, hepatocellular cancer, breast cancer, glioma, and so on. Although a range of targets and signaling pathways such as MACC1 and Wnt/ß-catenin signaling pathway were illustrated to be regulated by miRNA-338, which functions in tumor progression are still ambiguous and the underlying molecular mechanisms are also unclear. Herein, we reviewed the latest studies in miRNA-338 and summarized its roles in different type of human tumors, which might provide us new idea for further investigations and potential targeted therapy.

Cell-free exosome-laden scaffolds for tissue repair.

With the development of regenerative medicine, tissue repair at the molecular, cellular, tissue, and organ level has seen continuous improvements over traditional techniques. As the core of tissue repair, seed cells are widely used in various fields of regenerative medicine. However, their use is still associated with problems such as decreased cell survival and regeneration capacity after transplantation, immune rejection, and ethical concerns. Therefore, it is difficult to universally and safely apply stem cell banks for regenerative medicine. The paracrine effects of cells, especially secretion of exosomes, play vital roles in cell communication, immune response, angiogenesis, scar formation, tissue repair, and other biological functions. Exosomes are a type of nanoscale extracellular vesicle that contain biologically active molecules such as RNA and proteins; therefore, exosomes can replicate the functions of their parental cells. Meanwhile, exosomes can be used as nanocarriers to deliver active factors or small molecules to promote tissue repair. Preclinical studies of exosomes in tissue engineering and regenerative medicine have been carried in the fields of bone/cartilage repair, nerve regeneration, liver and kidney regeneration, skin repair, vascular tissue regeneration, etc. This review introduces exosomes from the aspects of biogenesis, composition, identification, and isolation, and focuses on the development status of scaffold materials for exosome delivery. In addition, we highlight examples of exosome-laden scaffolds for preclinical applications in tissue repair. We look forward to the broad application prospects of exosome-laden scaffolds.

3D printed gelatin/hydroxyapatite scaffolds for stem cell chondrogenic differentiation and articular cartilage repair.

Acute injury of the articular cartilage can lead to chronic disabling conditions because of the limited self-repair capability of the cartilage. Implantation of stem cells at the injury site is a viable treatment, but requires a scaffold with a precisely controlled geometry and porosity in the 3D space, high biocompatibility, and the capability of promoting chondrogenic differentiation of the implanted stem cells. Here we report the development of gelatin/hydroxyapatite (HAP) hybrid materials by microextrusion 3D bioprinting and enzymatic cross-linking as the scaffold for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The scaffold supports the adhesion, growth, and proliferation of hUCB-MSCs and induces their chondrogenic differentiation in vitro. Doping HAP in the gelatin scaffold increases the fluidity of the hydrogel, improves the gelation kinetics and the rheological properties, and allows better control over 3D printing. Implanting the hUCB-MSC-laden scaffold at the injury site of the articular cartilage effectively repairs the cartilage defects in a pig model. Altogether, this work demonstrates the 3D printing of gelatin-based scaffold materials for hUCB-MSCs to repair cartilage defects as a potential treatment of articular cartilage injury.

Forecasting sensitive targets of the kynurenine pathway in pancreatic adenocarcinoma using mathematical modeling.

In this study, a new mathematical model was established and validated to forecast and define sensitive targets in the kynurenine pathway (Kynp) in pancreatic adenocarcinoma (PDAC). Using the Panc-1 cell line, genetic profiles of Kynp molecules were tested. qPCR data were implemented in the algorithm programming (fmincon and lsqnonlin function) to estimate 35 parameters of Kynp variables by Matlab 2017b. All tested parameters were defined as non-negative and bounded. Then, based on experimental data, the function of the fmincon equation was employed to estimate the approximate range of each parameter. These calculations were confirmed by qPCR and Western blot. The correlation coefficient (R) between model simulation and experimental data (72 hours, in intervals of 6 hours) of every variable was >0.988. The analysis of reliability and predictive accuracy depending on qPCR and Western blot data showed high predictive accuracy of the model; R was >0.988. Using the model calculations, kynurenine (x3, a6), GPR35 (x4, a8), NF-kßp105 (x7, a16), and NF-kßp65 (x8, a18) were recognized as sensitive targets in the Kynp. These predicted targets were confirmed by testing gene and protein expression responses. Therefore, this study provides new interdisciplinary evidence for Kynp-sensitive targets in the treatment of PDAC.

Expression of LRRC8A is elevated in the cytoplasm of osteosarcoma tissues: An immunohistochemical study with tissue microarrays.

The purpose of the present study was to investigate the expression profile of leucine-rich repeat-containing protein 8A (LRRC8A) in osteosarcoma and normal cortical bone, as well as its association with sex, age and tumor malignancy. Immunohistochemical staining of osteosarcoma tissue microarrays (TMAs) was performed to determine the protein expression of LRRC8A and compare them among different subgroups. The expression of LRRC8A in the nuclei and cytoplasm of U2OS tumor cells and MC3T3-E1 osteoblast-like cells was determined using reverse transcription-quantitative PCR. Of all samples of the TMA for patients with osteosarcoma that were tested, 94% featured high cytoplasmic expression of LRRC8A, while in all normal bone tissue control groups, the gene was mainly expressed in the nucleus. In MC3T3-E1 osteoblasts, the expression of LRRC8A at the RNA level was mainly in the cytoplasm. The difference in expression of LRRC8A between microarrays and osteoblasts was statistically significant. In U2OS osteosarcoma cells, LRRC8A mRNA was concentrated in the nuclei and cytoplasm. In osteosarcoma, the expression level of LRRC8A was not significantly associated with sex or age. In conclusion, LRRC8A was highly expressed in the cytoplasm of osteosarcoma cells and the degree of expression may be associated with the degree of tumor malignancy.

The Arthroscopic Application of Radiofrequency in Treatment of Articular Cartilage Lesions.

Articular cartilage lesion is a common disease to be treated by arthroscopic surgery. It will eventually progress to osteoarthritis without proper management, which can affect patients' work and daily life seriously. Although mechanical debridement and laser have been used clinically for its treatment, due to their respective drawbacks, radiofrequency has drawn increasing attention from clinicians as a new technique with more advantages. However, the safety and efficacy of radiofrequency have also been questioned. In this article, the scope of application of radiofrequency was reviewed following an introduction of its development history and mechanism, and the methods to ensure the safety and effectiveness of radiofrequency through power and temperature control were summarized.

Pathological changes of frozen shoulder in rat model and the therapeutic effect of PPAR-γ agonist.

Frozen shoulder is a common shoulder disorder characterized by a gradual increase of pain and a limited range of motion. However, its pathophysiologic mechanisms remain unclear and there is no consensus as to the most effective treatment. The purpose of the study was to investigate the effect of transforming growth factor-ß (TGF-ß) on fibrosis and inflammatory response of the shoulder joint of rat models and to explore the therapeutic effect of the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist. In the study, the effect of PPAR-γ agonist CDDO-IM treatment on cell proliferation, migration, and extracellular matrix proteins synthesis (vimentin, α-smooth muscle actin, collagen I, and collagen III) were tested by cell proliferation test, scratches test, real-time quantitative polymerase chain reaction, and Western blot analysis. The frozen shoulder was also established on the rat model by injecting adenovirus-TGF-ß1 into rats' shoulder capsule. Pathological changes of the frozen shoulder tissue of the experimental group and PPAR-γ agonist treatment group were evaluated. The stiffness of joints of the three groups was tested. Inflammatory mediators' expression including cyclooxygenase-1, interleukin-1ß, and tumor necrosis factor-α of the shoulder was tested by enzyme-linked immunosorbent assay, and the expression of extracellular matrix proteins was evaluated by hematoxylin and eosin staining and immunohistochemistry. The results showed that pathological changes of the frozen shoulder in the rat model include an abnormal proliferation of fibroblasts, infiltration of inflammatory cells, and disorder of fibrous structure, while rosiglitazone reduced the severity of the frozen shoulder in the treatment group. Clinically, PPAR-γ agonists may be a promising target for the treatment of the frozen shoulder.

Exosome-mediated delivery of kartogenin for chondrogenesis of synovial fluid-derived mesenchymal stem cells and cartilage regeneration.

Transplantation of synovial fluid-derived mesenchymal stem cells (SF-MSCs) is a viable therapy for cartilage degeneration of osteoarthritis (OA). But controlling chondrogenic differentiation of the transplanted SF-MSCs in the joints remains a challenge. Kartogenin (KGN) is a small molecule that has been discovered to induce differentiation of SF-MSCs to chondrocytes both in vitro and in vivo. The clinical application of KGN however is limited by its low water solubility. KGN forms precipitates in the cell, resulting in low effective concentration and thus limiting its chondrogesis-promoting activity. Here we report that targeted delivery of KGN to SF-MSCs by engineered exosomes leads to even dispersion of KGN in the cytosol, increases its effective concentration in the cell, and strongly promotes the chondrogenesis of SF-MSCs in vitro and in vivo. Fusing an MSC-binding peptide E7 with the exosomal membrane protein Lamp 2b yields exosomes with E7 peptide displayed on the surface (E7-Exo) that has SF-MSC targeting capability. KGN delivered by E7-Exo efficiently enters SF-MSCs and induces higher degree of cartilage differentiation than KGN alone or KGN delivered by exosomes without E7. Co-administration of SF-MSCs with E7-Exo/KGN in the knee joints via intra-articular injection also shows more pronounced therapeutic effects in a rat OA model than KGN alone or KGN delivered by exosomes without E7. Altogether, transplantation of SF-MSCs with in situ chondrogenesis enabled by E7-Exo delivered KGN holds promise towards as an advanced stem cell therapy for OA.

Effects of ATP9A on Extracellular Vesicle Release and Exosomal Lipid Composition.

Numerous biological processes are regulated by the intercellular communications arising from extracellular vesicles (EVs) released from cells. However, the mechanisms that regulate the quantity of EV discharged have yet to be understood. While it is known that ATP9A, a P4-ATPase, is involved in endosomal recycling, it is not clear whether it also contributes to the release of EVs and the makeup of exosomal lipids. This study is aimed at exploring the role of human ATP9A in the process of EV release and, further, to analyze the profiles of EV lipids regulated by ATP9A. Our results demonstrate that ATP9A is located in both the intracellular compartments and the plasma membrane. The percentage of ceramides and sphingosine was found to be significantly greater in the control cells than in the ATP9A overexpression and ATP9A knockout groups. However, EV release was greater in ATP9A knockout cells, indicating that ATP9A inhibits the release of EVs. This study revealed the effects of ATP9A on the release of EVs and the lipid composition of exosomes.

Chondrocyte-Targeted MicroRNA Delivery by Engineered Exosomes toward a Cell-Free Osteoarthritis Therapy.

Targeted delivery to the diseased cell or tissue is the key to the successful clinical use of nucleic acid drugs. In particular, delivery of microRNA-140 (miRNA-140, miR-140) into chondrocytes across the dense, nonvascular extracellular matrix of cartilage remains a major challenge. Here, we report the chondrocyte-targeting exosomes as vehicles for the delivery of miR-140 into chondrocytes as a new treatment for osteoarthritis (OA). By fusing a chondrocyte-affinity peptide (CAP) with the lysosome-associated membrane glycoprotein 2b protein on the surface of exosomes, we acquire CAP-exosomes that can efficiently encapsulate miR-140, specifically enter, and deliver the cargo into chondrocytes in vitro. CAP-exosomes, in contrast to nontagged exosome vesicles, are retained in the joints after intra-articular injection with minimal diffusion in vivo. CAP-exosomes also deliver miR-140 to deep cartilage regions through the dense mesochondrium, inhibit cartilage-degrading proteases, and alleviate OA progression in a rat model, pointing toward a potential organelle-based, cell-free therapy of OA.

Functional Reconstruction of Hindfoot With Total Calcaneus and Talus Loss by Ilizarov Technique: A Case Report.

Total calcaneus and talus loss in the hindfoot is an unusual but severe condition encountered in clinical settings. This condition affects lower-extremity function and poses a significant challenge to limb salvage. We present a case of a 43-year-old man with total calcaneus and talus loss in the right foot treated by Ilizarov technique. A staged treatment protocol was planned to reconstruct and optimize the heel for weightbearing and walking. During the 15-month postoperative follow-up, the patient reported no significant discomfort in the targeted foot and regained satisfactory function, including shoe wearing, walking, driving, and climbing stairs. The American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Scale score was 71, which was an improvement from a preoperative score of 40. This case is the first reported on the functional reconstruction by Ilizarov technique of hindfoot with total calcaneus and talus loss. This treatment protocol provides an effective approach to reconstructing the hindfoot with massive bone loss, although the long-term outcome remains unknown.

Intra-articular injection of hUC-MSCs expressing miR-140-5p induces cartilage self-repairing in the rat osteoarthritis.

INTRODUCTION: Currently, osteoarthritis (OA) receives global increasing attention because it associates severe joint pain and serious disability. Stem cells intra-articular injection therapy showed a potential therapeutic superiority to reduce OA development and to improve treating outputs. However, the long-term effect of stem cells intra-articular injection on the cartilage regeneration remains unclear. Recently, miR-140-5p was confirmed as a critical positive regulator in chondrogenesis. We hypothesized that hUC-MSCs overexpressing miR-140-5p have better therapeutic effect on osteoarthritis. MATERIALS AND METHODS: To enhance stem cell chondrogenic differentiation, we have transfected human umbilical cord mesenchymal stem cells (hUC-MSCs) with miR-140-5p mimics and miR-140-5p lentivirus to overexpress miR-140-5p in a short term or a long term accordingly. Thereafter, MSCs proliferation, chondrogenic genes expression and extracellular matrix were assessed. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of SD rats as an OA model, and then intra-articular injection of hUC-MSCs or hUC-MSCs transfected with miR-140-5p lentivirus was carried to evaluate the cartilage healing effect with histological staining and OARSI scores. The localization of hUC-MSCs after intra-articular injection was further confirmed by immunohistochemical staining. RESULTS: Significant induction of chondrogenic differentiation in the miR-140-5p-hUC-MSCs (140-MSCs), while its proliferation was not influenced. Interestingly, intra-articular injection of 140-MSCs significantly enhanced articular cartilage self-repairing in comparison to normal hUC-MSCs. Moreover, we noticed that intra-articular injection of high 140-MSCs numbers reinforces cells assembling on the impaired cartilage surface and subsequently differentiated into chondrocytes. CONCLUSIONS: In conclusion, these results indicate therapeutic superiority of hUC-MSCs overexpressing miR-140-5p to treat OA using intra-articular injection.

Cell-to-Cell Culture Inhibits Dedifferentiation of Chondrocytes and Induces Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess great promise as a therapeutic to repair damaged cartilage. Direct intra-articular injection of mesenchymal stem cells has been shown to reduce cartilage damage and is advantageous as surgical implantation and associated side effects can be avoided using this approach. However, the efficacy of stem cell-based therapy for cartilage repair depends highly on the direct interactions of these stem cells with chondrocytes in the joint. In this study, we have carried out an in vitro cell-to-cell contact coculture study with human articular chondrocytes (hACs) and hUC-MSCs, with the goal of this study being to evaluate interactions between hACs and hUC-MSCs. METHODS: Low-density monolayer cultures of hUC-MSCs and hACs were mixed at a ratio of 1 : 1 in direct cell-to-cell contact groups. Results were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. RESULTS: A mixed coculture of hUC-MSCs and hACs was found to exhibit synergistic interactions with enhanced differentiation of hUC-MSCs and reduced dedifferentiation of chondrocytes. Mixed cultures after 21 days were found to exhibit sufficient chondrogenic induction. CONCLUSIONS: The results from this study suggest the presence of mutual effects between hUC-MSCs and hACs even culture at low density and provide further support for the use of intra-articular injection strategies for cartilage defect treatment.

Estrogen Modulates Cartilage and Subchondral Bone Remodeling in an Ovariectomized Rat Model of Postmenopausal Osteoarthritis.

BACKGROUND Estrogen levels regulate changes in osteoarthritis (OA) by inhibiting degradation of the extracellular matrix. Recent in vitro studies have also shown the role of microRNA-140-5p (miR-140-5p). This study aimed to investigate the role of estrogen deficiency, selective modulation of expression of the estrogen receptor (ER), and expression of miR-140-5p in cartilage and subchondral bone remodeling in an ovariectomized rat model of postmenopausal OA. MATERIAL AND METHODS Female Sprague-Dawley rats included two model groups, ovariectomized (OVX) rats and rats with destabilization of the medial meniscus (DMM) rats. Two months after surgery, estrogen levels were measured by the enzyme-linked immunosorbent assay (ELISA). Three-dimensional (3D) micro-computed tomography (micro-CT) was used to image the knee joints. Rats were treated with subcutaneous injection of estrogen (E2) or the selective estrogen receptor modulator (SERM), raloxifene (RAL), for one month. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect miR-140-5p in serum, and histology of the knee joint cartilage and bone was performed. RESULTS In the ovariectomized rat model of OA, estrogen therapy reduced the degree of cartilaginous degeneration, while treatment with raloxifene showed no significant effect. Expression levels of miR-140-5p in the OA model group were significantly lower than the control group. Micro-CT showed that in the model group, anterior cruciate ligament dislocation and subchondral bone density were significantly reduced. CONCLUSIONS In an ovariectomized rat model of postmenopausal OA, estrogen deficiency resulted in resorption of subchondral bone and degeneration of articular cartilage.

Repair of osteochondral defects using injectable chitosan-based hydrogel encapsulated synovial fluid-derived mesenchymal stem cells in a rabbit model.

The regeneration of hyaline articular cartilage remains a major challenge due to the limited potential for cartilage to self-repair. Mesenchymal stem cell and hydrogel scaffold-based cartilage tissue engineering is a promising technique for articular cartilage therapy. The purpose of this study was to investigate the use of rabbit synovial fluid mesenchymal stem cells (rbSF-MSCs) encapsulated in an injectable chitosan-based hydrogel to repair full-thickness cartilage defects in femoral patellar grooves in rabbits. The rbSF-MSCs were obtained from rabbit synovial fluid and the surface markers of rbSF-MSCs were coincidental to the identification criteria of MSCs according to flow cytometry. The rbSF-MSCs were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. In the present study, rbSF-MSCs encapsulated in glycol chitosan (GC) and benzaldehyde capped poly (ethylene oxide) (OHC-PEO-CHO) hydrogel were introduced into rabbits to repair articular cartilage defects. The modulus of the hydrogel could be regulated by the concentrations of GC and OHC-PEO-CHO and the hydrogel has a good biocompatibility to rbSF-MSCs. Assessment of in vivo repair indicates using hydrogel/rbSF-MSCs was superior to using the hydrogel scaffold only and the untreated control based on gross appearance and histological grading and evaluation. These preliminary findings suggest using the injectable chitosan-based hydrogel as a scaffold and rbSF-MSCs as seed cells is an alternative for tissue engineering of in vivo treatments for cartilage defects and these rbSF-MSCs allografts may be promising for use in clinical applications.

Pulse electromagnetic fields enhance the repair of rabbit articular cartilage defects with magnetic nano-hydrogel.

Hydrogel is an important scaffold material in regenerative medicine and cartilage tissue engineering. Hydrogel material combined with pulse electromagnetic fields (PEMFs), PEMFs has the potential to manage the repair of defective articular cartilage. Here, we developed a new type of magnetic hydrogel. The data shows that the magnetic hydrogel had good mechanical properties, and its surface had micropores and unevenness, which was conducive to cell adhesion growth. Infrared spectroscopy analysis showed that the magnetic particles were evenly distributed in the hydrogel, and the addition of constant static magnetic field yielded magnetic water. The hydrogel exhibited good superparamagnetism. The co-culture of the magnetic hydrogel and bone marrow mesenchymal stem cells (BMSCs) showed good biocompatibility. The PEMFs promoted the differentiation of the BMSCs into cartilage, and the index of cartilage differentiation increased obviously. The results of the animal experiments showed that the magnetic hydrogel and BMSCs combined with pulsed electromagnetic field had a strong repair effect. They also showed that the magnetic nano-hydrogel combined with the PEMFs induced chondrogenic differentiation of the BMSCs. The positive experimental results suggested that the combination of magnetic hydrogel and the PEMFs can be used as an effective method for repairing articular cartilage defects in rabbit model.

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.