Mechanisms of ferroptosis with immune infiltration and inflammatory response in rotator cuff injury.

The processes driving ferroptosis and rotator cuff (RC) inflammation are yet unknown. The mechanism of ferroptosis and inflammation involved in the development of RC tears was investigated. The Gene Expression Omnibus database was used to obtain the microarray data relevant to the RC tears for further investigation. In this study, we created an RC tears rat model for in vivo experimental validation. For the additional function enrichment analysis, 10 hub ferroptosis-related genes were chosen to construct the correlation regulation network. In RC tears, it was discovered that genes related to hub ferroptosis and hub inflammatory response were strongly correlated. The outcomes of in vivo tests showed that RC tears were related to Cd68-Cxcl13, Acsl4-Sat1, Acsl3-Eno3, Acsl3-Ccr7, and Ccr7-Eno3 pairings in regulating ferroptosis and inflammatory response. Thus, our results show an association between ferroptosis and inflammation, providing a new avenue to explore the clinical treatment of RC tears.

3D Printed Platelet-Rich Plasma-Loaded Scaffold with Sustained Cytokine Release for Bone Defect Repair.

The combination of three-dimensional (3D) printed scaffold materials and various cytokines can achieve the purpose of tissue reconstruction more efficiently. In this study, we prepared platelet-rich plasma (PRP)/gelatin microspheres combined with 3D printed polycaprolactone/ß-tricalcium phosphate scaffolds to solve the key problem that PRP cannot be released under control and the release time is too short, and thus better promote bone repair. Consequently, the composite scaffold displayed a good mechanical property and sustained cytokine release for ∼3 weeks. Increased survival, proliferation, migration, and osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells were observed compared with the control groups. The in vivo study demonstrated that the composite scaffold with PRP/gelatin microspheres led to greater positive effects in promoting large bone defect repair. In conclusion, in this study, a new type of PRP long-term sustained-release composite scaffold material was constructed that effectively improved the survival, proliferation, and differentiation of cells in the transplanted area, thereby better promoting the repair of large bone defects. Impact statement Reconstruction of bone tissue and blood vessels at bone defects takes time. Platelet-rich plasma (PRP) has been widely used in bone defect repair because it contains a variety of cytokine that can promote local osteogenesis and angiogenesis. In this study, we constructed a new type of polycaprolactone/ß-tricalcium phosphate/PRP/gelatin scaffold to solve the predicament of short cytokine release time in PRP-related materials. We proved that this scaffold can not only achieve long-term PRP-related cytokine release (more than 3 weeks) but also promote osteogenesis and bone defect repair. We believe that this is a novel concept of developing the sustained PRP-related cytokine releasing bioscaffold for treating large bone defect.

3D printed polycaprolactone/beta-tricalcium phosphate/magnesium peroxide oxygen releasing scaffold enhances osteogenesis and implanted BMSCs survival in repairing the large bone defect.

Ischemia and hypoxia in the bone defect area remain an intractable problem when treating large bone defects. Thus, oxygen-releasing biomaterials have been widely researched in recent years. Magnesium peroxide (MgO2) can release oxygen (O2), and magnesium ions (Mg2+), simultaneously, which is seen to have significant potential in bone substitutes. In this study, we used 3D printing technology to fabricate a MgO2-contained composite scaffold, which was composed of polycaprolactone (PCL), beta-tricalcium phosphate (ß-TCP) and magnesium peroxide (MgO2). Physical properties and O2/Mg2+ releasing behavior of the scaffold were studied. Then, we evaluated the effects of the scaffold on cell survival, proliferation, migration, adhesion and osteogenic differentiation by the co-culture of bone marrow mesenchymal stem cells (BMSCs) and scaffold under normoxia and hypoxia in vitro. Finally, the osteogenic properties of the scaffold in vivo were evaluated via the rat femoral condylar bone defect model. The PCL/ß-TCP/MgO2 scaffold showed good mechanical properties and sustained O2 and Mg2+ release for about three weeks. Meanwhile, the scaffold showed appreciable promotion on the survival, proliferation, migration and osteogenic differentiation of BMSCs under hypoxia compared with control groups. The results of imaging studies and histological analysis showed that implantation of PCL/ß-TCP/MgO2 scaffold could promote seed cell survival and significantly increased new bone formation. In sum, the PCL/ß-TCP/MgO2 scaffold is promising with great potential for treating large bone defects.

CaO2/gelatin oxygen slow-releasing microspheres facilitate tissue engineering efficiency for the osteonecrosis of femoral head by enhancing the angiogenesis and survival of grafted bone marrow mesenchymal stem cells.

The osteonecrosis of femoral head (ONFH), a common refractory disease, is still not fully understood today. Hypoxia caused by ischemia is not only an important pathogenic factor but also a critical challenge for the survival of seed cells in the tissue engineering therapy of ONFH. To explore an efficient strategy to treat ONFH by targeting hypoxia, newly designed CaO2/gelatin microspheres were composited with 3D printed polycaprolactone/nano-hydroxyapatite (PCL/nHA) porous scaffold, sodium alginate/gelatin hydrogel, and bone marrow mesenchymal stem cells (BMSCs) to develop a novel tissue engineering scaffold and then transplanted into the core depression area of the ONFH rabbit model. The current data demonstrated that CaO2/gelatin microspheres can constantly release oxygen for 19 days. In vitro assays with BMSCs illustrated that scaffolds have high biocompatibility and are favorable for cell proliferation in extreme hypoxia (1% O2). The in vivo study demonstrated that the transplanted scaffold with oxygen-generating microspheres significantly enhanced the osteogenic and angiogenic effects compared to the scaffold without microspheres. Further assessments revealed that microspheres in the scaffold can reduce the local cell apoptosis and enhance the survival of grafted cells in the host. Collectively, the present study developed a novel oxygen slow-releasing composite scaffold, which can facilitate tissue engineering efficiency for treating the osteonecrosis of the femoral head by enhancing the angiogenesis and survival of grafted stem cells.

Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (ONFH) is characterized by decreased osteogenesis, angiogenesis, and increased adipogenesis. While bone tissue engineering has been widely investigated to treat ONFH, its therapeutic effects remain unsatisfactory. Therefore, further studies are required to determine optimal osteogenesis, angiogenesis and adipogenesis in the necrotic area of the femoral head. In our study, we developed a carboxymethyl chitosan/alginate/bone marrow mesenchymal stem cell/endothelial progenitor cell (CMC/ALG/BMSC/EPC) composite implant, and evaluated its ability to repair steroid-induced ONFH. Our in vitro studies showed that BMSC and EPC coculture displayed enhanced osteogenic and angiogenic differentiation. When compared with single BMSC cultures, adipogenic differentiation in coculture systems was reduced. We also fabricated a three-dimensional (3D) CMC/ALG scaffold for loading cells, using a lyophilization approach, and confirmed its good cell compatibility characteristics, that is, high porosity, low cytotoxicity and favorable cell adhesion. 3D coculture of BMSCs and EPCs also promoted secretion of osteogenic and angiogenic factors. Then, we established an rabbit model of steroid-induced ONFH. The CMC/ALG/BMSC/EPC composite implant was transplanted into the bone tunnel of the rabbit femoral head after core decompression (CD) surgery. Twelve weeks later, radiographical and histological analyses revealed CMC/ALG/BMSC/EPC composite implants had facilitated the repair of steroid-induced ONFH, by promoting osteogenesis and angiogenesis, and reducing adipogenesis when compared with CD, CMC/ALG, CMC/ALG/BMSC and CMC/ALG/EPC groups. Thus, our data show that cotransplantation of BMSCs and EPCs in 3D scaffolds is beneficial in treating steroid-induced ONFH.

The Regulatory Effect of VEGF-Ax on Rat Bone Marrow Mesenchymal Stem Cells' Angioblastic Differentiation and Its Proangiogenic Ability.

Vascular endothelial growth factor A (VEGFA), which plays a key role in angiogenesis, is composed of many isoforms. Distinct VEGFA isoforms are generated by alternative splicing of VEGFA mRNA and named as VEGFxxx, where xxx represents the number of amino acids present in the final protein sequence. These isoforms have opponent pro- and antiangiogenic effects. VEGF-Ax, an additional isoform containing a 22-amino-acid extension in the COOH terminus, arising from VEGFA mRNA, programmed translational readthrough. The function of VEGF-Ax is not clear, especially the conclusion that VEGF-Ax regulates angiogenesis is contradictory. Thus, we investigated the effect of VEGF-Ax on differentiation and angiogenesis of rat bone marrow mesenchymal stem cells (BMMSCs). The results showed that VEGF-Ax could promote the proliferation and migration of BMMSCs, stimulate the differentiation of BMMSCs into endothelial cell-like cells, and protect BMMSCs from endoplasmic reticulum stress-induced apoptosis.

PC-3-Derived Exosomes Inhibit Osteoclast Differentiation by Downregulating miR-214 and Blocking NF-κB Signaling Pathway.

Prostate cancer is a serious disease that can invade bone tissues. These bone metastases can greatly decrease a patient's quality of life, pose a financial burden, and even result in death. In recent years, tumor cell-secreted microvesicles have been identified and proposed to be a key factor in cell interaction. However, the impact of cancer-derived exosomes on bone cells remains unclear. Herein, we isolated exosomes from prostate cancer cell line PC-3 and investigated their effects on human osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining. The potential mechanism was evaluated by qRT-PCR, western blotting, and microRNA transfection experiments. The results showed that PC-3-derived exosomes dramatically inhibited osteoclast differentiation. Marker genes of mature osteoclasts, including CTSK, NFATc1, ACP5, and miR-214, were all downregulated in the presence of PC-3 exosomes. Furthermore, transfection experiments showed that miR-214 downregulation severely impaired osteoclast differentiation, whereas overexpression of miR-214 promoted differentiation. Furthermore, we demonstrated that PC-3-derived exosomes block the NF-κB signaling pathway. Our study suggested that PC-3-derived exosomes inhibit osteoclast differentiation by downregulating miR-214 and blocking the NF-κB signaling pathway. Therefore, elevating miR-214 levels in the bone metastatic site may attenuate the invasion of prostate cancer.

Effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of MC3T3-E1 cells.

In physiological and pathological environments, the concentration of oxygen around osteoblasts varies widely. No studies have systematically evaluated the effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of osteoblasts. In this study, we cultured the osteoblast precursor cell line MC3T3-E1 in small individual chambers with oxygen concentrations of 1%, 3%, 6%, 9%, and 21%. Cell proliferation was evaluated by the proliferation index test and EdU staining. To test cell survival, a live/dead assay was performed. A tablet scratch assay was performed to detect the migratory ability of the cells. Bone nodule formation experiments and immunofluorescence and Western blotting analyses of osteogenic-related proteins were performed to assess the osteogenic differentiation of the cells. We found that the proliferation and osteogenic differentiation ability of MC3T3-E1 cells in different oxygen concentrations were both approximately bell-shaped curves and that the optimal oxygen concentrations were approximately 6% and 9%, respectively. The live/dead assay showed that the survival of MC3T3-E1 cells in different oxygen concentrations was affected by the amount of serum. The tablet scratch experiment showed that there was greater cell migration with oxygen concentrations of 1%, 3%, and 21% than with oxygen concentrations of 6% and 9%. Our results have significant reference value for the intervention of the pathological processes involving osteoblasts, such as fracture, osteoporosis, and some vascular diseases. These results also have an important guiding role for the new scientific idea that osteoblasts can function as treatment cells to repair bone defects.

Self-assembling peptide and nHA/CTS composite scaffolds promote bone regeneration through increasing seed cell adhesion.

Porous scaffolds fabricated with nano-hydroxyapatite (nHA) and chitosan (CTS), are widely used in bone tissue engineering (BTE). However, cell adhesion is relatively poor in nHA/CTS scaffolds, which also do not provide an ideal three-dimensional environment for seed cells. These deficiencies limit the applicability of these BTE scaffolds to repair bone defects. To address these challenges, we designed a composite scaffold that combines nHA/CTS with self-assembling peptide (SAP), a material which is similar to the extracellular matrix. We found that SAP/nHA/CTS scaffolds both increased the adhesion of bone mesenchymal stem cells (BMSCs) and enhanced the mechanical properties of the scaffold. This composite scaffold was then used to repair a femoral condylar bone defect in a mouse model. Healing and mineralization was demonstrated after 12 weeks using H&E staining, microcomputerized tomography, and bone mineral density tests. To our knowledge, this is the first report that SAP/nHA/CTS scaffolds can increase cell adhesion and promote the reconstruction of femoral condylar bone defects. Moreover, this study indicates that BTE using a SAP/nHA/CTS scaffold may be a novel prospective strategy for healing extensive bone defects.