CRISPR/Cas12a and aptamer-chemiluminescence based analysis for the relative abundance determination of tumor-related protein positive exosomes for breast cancer diagnosis.

Exosomes, as novel biomarker for liquid biopsy, exhibit huge important potential value for cancer diagnosis. However, various proteins show different expression levels on exosomal membrane, and the absolute concentration of exosomes in clinical samples is easily influenced by a number of factors. Here, we developed a CRISPR/Cas12a and aptamer-chemiluminescence based analysis (CACBA) for the relative abundance determination of tumor-related protein positive exosomes in plasma for breast cancer diagnosis. The total concentration of exosomes was determined through captured CD63 using a CRISPR/Cas12a-based method with the LoD of 8.97 × 103 particles/µl. Meanwhile, EpCAM and MUC1 positive exosomes were quantitatively detected by aptamer-chemiluminescence (ACL) based method with the LoD of 1.45 × 102 and 3.73 × 102 particles/µl, respectively. It showed that the percentages of EpCAM and MUC1 positive exosomes offered an excellent capability to differentiate breast cancer patients and healthy donors. The high sensitivity, strong specificity, outstanding anti-interference capability, and steady recovery rate of this approach offered higher accuracy and robustness than the commercialized method in clinical trial. In addition with good stability, easy preparation and low cost, this method not only provides a new approach to rapid analysis of exosome proteins, it may be quickly extended to the diagnoses of various cancers.

Exosomes loaded with chondrogenic stimuli agents combined with 3D bioprinting hydrogel in the treatment of osteoarthritis and cartilage degeneration.

Osteoarthritis (OA) is a challenging joint inflammatory disease that often leads to disability. Immunoregulatory Exosomes (Exos) have shown promise in OA and cartilage degeneration treatment. Engineering Exos to deliver therapeutic agents like Kartogenin (KGN) has displayed potential for restoring cartilage regeneration. However, challenges include the uneven distribution of Exos at the injury site and the release of Exos cargo out of chondrocytes. Hydrogel-loaded uMSC-Exo has demonstrated significant therapeutic effects in wound healing and tissue regeneration. Recently, a new version of three-dimensional (3D) bioprinting of hydrogel significantly restored cartilage regeneration in OA joints. Combining immune regulatory Exos with 3D bioprinting hydrogel (3D-BPH-Exos) holds the potential for immunomodulating cartilage tissue and treatment of OA. It can reduce intracellular inflammasome formation and the release of inflammatory agents like IL-1ß, TNF-α, and INF-γ, while also preventing chondrocyte apoptosis by restoring mitochondrial functions and enhancing chondrogenesis in synovial MSCs, osteoprogenitor cells, and osteoclasts. Loading Exos with chondrogenic stimuli agents in the 3D-BPH-Exos approach may offer a faster and safer strategy for cartilage repair while better inhibiting joint inflammation than high doses of anti-inflammatory drugs and cell-based therapies. This review provides a comprehensive overview of hydrogel bioprinting and exosome-based therapy in OA. It emphasizes the potential of 3D-BPH-Exos loaded with chondrogenic stimuli agents for OA treatment, serving as a basis for further research.

[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.].

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.

Insights into the implementation of Fibronectin 1 in the cartilage tissue engineering.

Recently, cartilage tissue engineering has become a cornerstone to treat cartilage degeneration and osteoarthritis (OA). Fibronectin1 (FN1) is described as multiple functional glycoproteins that play an essential role in chondrogenic and osteogenic differentiation. Few studies reported the potential of FN1 to enhance tissue engineering and reduce the death of chondrocytes in OA. Further, FN1 possesses multiple binding domains including collagen, integrin, and heparin that can interact with heparan sulfate proteoglycans at the surface of chondrocyte leading to promote cell signaling and differentiation. Recent studies suggested that FN1 can promote chondrocyte differentiation by upregulating TGF-ß/PI3K/Akt pathways. Further, FN1 can inhibit the apoptosis of chondrocytes by preventing the release of metalloproteinases through lowering the expression of p-PI3K/PI3K and p-AKT/AKT pathways. However, the use of FN1 in cartilage repair studies using animal models or clinical trials was rarely reported. Therefore, this article provides new insights into the importance of FN1 in cartilage tissue engineering to encourage more studies concerning FN1 in cartilage repair studies. Further, we provided new suggestions for advanced applications of FN1 to treat OA and cartilage degeneration.

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.

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.

MicroRNA-124 acts as a tumor-suppressive miRNA by inhibiting the expression of Snail2 in osteosarcoma.

The aim of the present study was to investigate the clinical significance of hsa-microRNA-124-3p (miR-124) in osteosarcoma (OS), and examine its role in cell growth and invasion. Using a microRNA chip array, the expression of miR-124 was detected in samples of surgically resected OS and matched against the levels of expression in tumor-adjacent normal tissues. The levels of miR-124 were upregulated in the OS cells through the transfection of miR-124 mimics. Cell proliferation and Transwell assays were performed to determine cell proliferation and invasion; Reverse transcription-quantitative polymerase chain reaction, western blot and luciferase assays were then used to detect the expression of the target gene snail family zinc finger 2 (Snail2). The expression of miR-124 was significantly lower in the OS tissues, compared with that in the tumor-adjacent normal tissues; and the expression of miR-124 in the tumor tissues was significantly associated with tumor size. miR-124 directly repressed the expression of Snail2, and resulted in a significant inhibition of cell proliferation and invasion. In a mouse model, the overexpression of miR-124 significantly inhibited U2OS cell proliferation and invasion. Taken together, miR-124 was associated with the adverse clinical and pathological features observed in OS. It acted as a tumor suppressor to regulate the proliferation and invasion of OS cells by targeting Snail2, suggesting that miR-124 may be key in the progression of OS.

Preparation and biocompatibility of diphasic magnetic nanocomposite scaffold.

We describe the study of a new type of diphasic magnetic nanocomposite scaffold (PLGA/Col-I-PLGA/n-HA/Fe2O3) and its preparation using a novel low-temperature deposition manufacturing (LDM) technology. In order to study the biocompatibility of this scaffold, we evaluated and explored its feasibility as a scaffold for tissue engineering. Diphasic magnetic nanocomposite scaffolds (PLGA/Col-I-PLGA/n-HA/Fe2O3) were prepared using LDM technology. The mechanical properties of the scaffold were tested using an electronic testing machine, electron microscopy was utilized to observe the ultrastructure, and a medium (ethanol) immersion method was used to determine the porosity of the scaffold. The scaffold was co-cultured with bone mesenchymal stem cells (BMSCs) and was induced to differentiate. The biocompatibility of the scaffold was then tested. The mechanical test results of the diphasic magnetic nanocomposite scaffold demonstrated good mechanical properties. Electron microscopy studies revealed two layers of pore sizes each with a uniform distribution, with the upper cartilage pore size observed to be small while the middle continuous phase was found to be in a good integration. Pore size and porosity test results demonstrated a cartilage layer pore size of 186 µm, with a porosity measured to be 89.5%. The pore size and porosity of the bone layer were 394 µm and 86.1%, respectively. These properties met the design requirements of double layer scaffolds. Co-culture of the diphasic magnetic nanocomposite scaffold and bone mesenchymal stem cells (BMSCs) exhibited good proliferation of bone mesenchymal stem cells (BMSCs), and the scaffold was found to be able to promote differentiation of the differentiation-oriented cells. These results demonstrated a good biocompatibility of the diphasic magnetic nanocomposite scaffold. The diphasic magnetic nanocomposite scaffold (PLGA/Col-I-PLGA/n-HA/Fe2O3) was found to have suitable mechanical properties as well as cell compatibility. The measured pore size and porosity met the requirements for cell adhesion and cell growth, which matched more closely to that of the physiological structure of normal articular cartilage and subchondral bones. We expect this to represent new technology for improved repair of cartilage and subchondral bone lesions caused by osteoarthritis or trauma.

Osteogenic differentiation of bone marrow mesenchymal stem cells by magnetic nanoparticle composite scaffolds under a pulsed electromagnetic field.

This study was conducted to investigate the effect of magnetic nanoparticle composite scaffold under a pulsed electromagnetic field on bone marrow mesenchymal stem cells (BMSCs), which was achieved by examining the biological behaviors of cell adhesion, proliferation and differentiation on the surface of the scaffolds. This may provide some experimental evidence for the use of magnetic nanoparticles in medical application. The magnetic nanoparticle composite scaffolds were evaluated and characterized by the following indexes: the cell proliferation was detected by the CCK-8 method, the alkaline phosphatase (ALP) activity was examined by a detection kit, and the expression of type I collagen and osteocalcin gene were evaluated by RT-PCR. The CCK-8 test showed that there was no significant difference in Group A (BMSCs-seeded magnetic scaffolds under the electromagnetic field), B (BMSCs-seeded magnetic scaffolds) and C (BMSCs cultured alone) (P > 0.05). The value for the ALP activity in Group A was higher than the other two groups. In addition, the RT-PCR results showed that the expression of type I collagen gene in Group A was enhanced (P < 0.05), suggesting that the magnetic nanoparticles combined with the pulsed electromagnetic field had a positive effect on the osteogenic differentiation of BMSCs. However, the expression of osteocalcin was not significantly different in three groups (P > 0.05). To conclude, magnetic nanoparticles may induce the osteogenic differentiation with the action of the pulsed electromagnetic field.

DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro.

DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

Effect of inorganic/organic ratio and chemical coupling on the performance of porous silica/chitosan hybrid scaffolds.

Inorganic/organic hybrid scaffolds have great potential for tissue engineering applications due to controllable mechanical properties and tailorable biodegradation. Here, silica/chitosan hybrid scaffolds were fabricated through the sol-gel method with a freeze drying process. 3-Glycidoxypropyl trimethoxysilane (GPTMS) and tetraethylorthosilicate (TEOS) were used as the covalent inorganic/organic coupling agent and the separate inorganic source, respectively. Hybrid scaffolds with various inorganic/organic weight ratios (I/Os) and molar ratios of chitosan and GPTMS (GCs) were examined and compared in this study. FTIR showed that higher GPTMS content resulted in the increased covalent cross-linking of the chitosan and the silica network in hybrids. Compression testing indicated that increasing the GPTMS content greatly improved the compressive strength of scaffold. LIVE/DEAD assay showed that enhanced cytocompatibility was obtained as the silica content increased. Therefore, the results confirmed that the two parameters I/O and GC can largely influence the scaffold performance, which can be used to tailor the hybrid properties.

Cytocompatibility of PLA/Nano-HA composites for interface fixation.

OBJECTIVE: When preliminary tests have confirmed a nano-hydroxyapatite (Nano-HA) content of 20% of the polylactic acid (PLA) composite material of Nano-HA interface fixation material for biomechanical requirements, there is a need for further observation of its biocompatibility and clinical applications, to provide reference data. METHODS: Preparation of Nano-HA content of 20% PLA composite Nano-HA bone substitute material and extract. The establishment of the negative control group (containing 10% fetal bovine serum in DMEM complete medium), experimental group (extract), the positive control group (mass concentration of 0.64% phenol), and a co-culture of rabbit bone marrow mesenchymal stem cells (rBMSC) and materials extraction liquid. Observation of the morphological changes in rBMSC in culture at time points of 3, 5, and 7 days, the use of the MTT assay, and determination of the relative growth in the above set of rBMSC in cell culture at 3, 5, and 7 days, to judge the material's cytotoxicity. RESULTS: With time, the absorbance value of the three groups of cells were significantly increased (P < 0.01). The relative growth of the rBMSCs in experimental group in the first 3, 5, and 7 days was 95.3%, 96.8% and 97.6%; the cytotoxicity was according to the national standards I; the difference was not significant (P > 0.05) between the the experimental group and the negative control group; there was a significant difference between the positive control group and the other 2 groups (P < 0.05). Cells in the experimental group were seen having normal morphology, and spindle-shaped adherent growth. CONCLUSION: PLA composite artificial bone materials and Nano-HA show good cell compatibility, and the values for cytotoxicity, with reference to GB/T16886.5.2003 (China) standards, are in the safe range.

MicroRNA-33a-5p suppresses growth of osteosarcoma cells and is downregulated in human osteosarcoma.

A body of evidence has indicated that microRNAs (miRNAs) may have significant roles in cancer. Aberrant expression of miRNAs has frequently been observed in various human malignancies, including osteosarcoma (OS). However, the roles of miRNAs in OS remain poorly understood. In the present study, high-throughput deep sequencing was performed to screen for deregulated miRNAs in OS. Screening identified 310 miRNAs which were significantly overexpressed and 41 miRNAs which were significantly downregulated (>2-fold) in OS samples, compared with adjacent non-tumor bone tissues. Among these miRNAs, miR-33a-5p was notably downregulated. TaqMan reverse transcription-polymerase chain reaction analysis further verified that miR-33a-5p expression was significantly reduced in a large cohort of human OS samples. Enhancing miR-33a-5p expression via transfection with miR-33a-5p precursor significantly inhibited OS cell growth, suggesting potential antitumor properties of miR-33a-5p. The results of the present study provide novel insights into the miRNAs involved in OS, and suggest that miR-33a-5p may function as a tumor suppressor in OS. Therefore, miR-33a-5p may be able to serve as a diagnostic and therapeutic target for OS treatment.

Study on clinical application of nano-hydroxyapatite bone in bone defect repair.

OBJECTIVE: To study the clinical effect of bone defect treated with nano-hydroxyapatite(Nano-HA) artificial bone. METHODS: From September 2009 to June 2012, 27 cases of bone defect were analyzed retrospectively. The position of bone defect included humerus, radius, ulna, femur, tibia and calcaneus. The range of bone defect was from 0.3 × 1.0 cm to 3 × 6.5 cm. Among them, there were 22 cases with fractures and 5 cases with tumors. All patients were treated with Nano-HA artificial bone. The ability of bone defect repair was evaluated by X-ray exams performed preoperatively and postoperatively. HSS scores were adopted for final evaluation at the latest follow-up. RESULTS: The patients were followed up from 11 to 26 months (average of 18.5 months). No general side effects occurred. X-ray photo showed an integrity interface between Nano-HA and bone. Primary healing was obtained in all cases without any complication. CONCLUSION: The Nano-HA artificial bone had a good biocompatibility and could be an ideal artificial bone in the reconstruction of bone defect.

[Study on the technological process of extraction and clathration for Rhizoma Atractylodis Macrocephalae oil with beta-cyclodextrin].

OBJECTIVE: To study the optimal extraction and clathration technology of Rhizoma Atractylodis Macrocephalae oil in Heweilichang Pill. METHODS: Orthogonal test was employed for selecting the optimum of extraction technology of Rhizoma Atractylodis Macrocephalae oil and the oil extraction rate were used as a index. The optimum including technology was chosen by determining the oil-bearing rate and extract ratio of inclusion compound. The inclusion compound were identified with thin chromatogram. RESULTS: The extraction technology was 10 volumes of water, extracted 6h with thick granula of Rhizoma Atractylodis Macrocephalae. The optimum preparation conditions for clathrate were established as beta-CD: oil was 6: 1, 3. 5 times of water, triturated for 75 minutes. CONCLUSION: The process is feasible and the method can be used for production.