Circ-ZNF236 mediates stem cells from apical papilla differentiation by regulating LGR4-induced autophagy.

AIM: Human stem cells from the apical papilla (SCAPs) are an appealing stem cell source for tissue regeneration engineering. Circular RNAs (circRNAs) are known to exert pivotal regulatory functions in various cell differentiation processes, including osteogenesis of mesenchymal stem cells. However, few studies have shown the potential mechanism of circRNAs in the odonto/osteogenic differentiation of SCAPs. Herein, we identified a novel circRNA, circ-ZNF236 (hsa_circ_0000857) and found that it was remarkably upregulated during the SCAPs committed differentiation. Thus, in this study, we showed the significance of circ-ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms. METHODOLOGY: The circular structure of circ-ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK-8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT-PCR, Alkaline phosphatase (ALP) and Alizarin red staining (ARS) were performed to explore the regulatory effect of circ-ZNF236/miR-218-5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs in vitro. Fluorescence in situ hybridization, as well as dual-luciferase reporting assays, revealed that circ-ZNF236 binds to miR-218-5p. Transmission electron microscopy (TEM) and mRFP-GFP-LC3 lentivirus were performed to detect the activation of autophagy. RESULTS: Circ-ZNF236 was identified as a highly stable circRNA with a covalent closed loop structure. Circ-ZNF236 had no detectable influence on cell proliferation but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ-ZNF236 was confirmed as a sponge of miR-218-5p in SCAPs, while miR-218-5p targets LGR4 mRNA at its 3'-UTR. Subsequent rescue experiments revealed that circ-ZNF236 regulates odonto/osteogenic differentiation by miR-218-5p/LGR4 in SCAPs. Importantly, circ-ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs. Subsequently, in vivo experiments showed that SCAPs overexpressing circ-ZNF236 promoted bone formation in a rat skull defect model. CONCLUSIONS: Circ-ZNF236 could activate autophagy through increasing LGR4 expression, thus positively regulating SCAPs odonto/osteogenic differentiation. Our findings suggested that circ-ZNF236 might represent a novel therapeutic target to prompt the odonto/osteogenic differentiation of SCAPs.

H2O2-Responsive Polymeric Micelles of Biodegradable Aliphatic Poly(carbonate)s as Promising Therapeutic Agents for Inflammatory Diseases.

Excessive amounts of reactive oxygen species (ROS) cause various biological damages and are involved in many diseases, such as cancer, inflammatory and thrombotic complications, and neurodegenerative diseases. Thus, ROS-responsive polymers with inherent ROS scavenging activity and biodegradability are extremely needed for the efficient treatment of ROS-related diseases. Here, this work fabricates the amphiphilic diblock copolymer PEG-b-PBC via ring-opening polymerization (ROP) of phenylboronic acid ester conjugated cyclic carbonate monomer. The copolymer easily forms micelles (BCM) and scavenges ROS rapidly. BCM not only releases the delivered drug but degrades to produce the small molecules p-hydroxybenzyl alcohol (HBA) with anti-inflammatory capability in the presence of H2O2. BCM can reduce the oxidative stress of human umbilical vein endothelial cells (HUVEC) and the levels of inflammatory factors secreted by macrophages, showing antioxidative and anti-inflammatory activity. Finally, BCM exerts a significant capability to reduce the complications of inflammation and thrombosis in vivo. The biodegradable aliphatic poly(carbonate)s have the potential to be used for drug delivery systems (DDS) for diseases induced by reactive oxygen species.

ROS-responsive phenylboronic ester-based nanovesicles as multifunctional drug delivery systems for the treatment of inflammatory and thrombotic complications.

Inflammatory and thrombotic complications and a low loading of dual drugs with different hydrophilicities remain challenges to treat thrombosis with drug delivery systems (DDSs). Here, the reactive oxygen species (ROS)-responsive amphiphilic block polymer poly(ethylene glycol)-b-2-((((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)carbonyl)oxy)-ethyl methacrylate (PEG-b-PTBEM) was synthesized and nanovesicles (PPTV) were prepared successfully for the drug delivery platform by controlling the hydrophilic/hydrophobic ratio of molecular chains and molecular self-assembly. The anti-inflammatory drug indomethacin (IDM) was loaded in the wall of nanovesicles and the thrombolytic enzyme nattokinase (NK) was encapsulated in the aqueous cavity of nanovesicles. Both drugs could be rapidly released at the site of thrombosis and/or inflammation with an excessive ROS concentration. The dual drug-loaded nanovesicles not only eliminated ROS, but also alleviated inflammation and dissolved the generated thrombus, showing significant therapeutic efficacy in the in vivo mouse model of carrageenan tail thrombosis. Therefore, drug-delivery nanovesicles play multiple roles in the treatment of inflammation-induced thrombotic disorders, which offer a promising treatment for inflammatory and thrombotic complications.

Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells.

Bone defects caused by bone trauma, infection, surgery, or other systemic diseases remain a severe challenge for the medical field. To address this clinical problem, different hydrogels were exploited to promote bone tissue regrowth and regeneration. Keratins are natural fibrous proteins found in wool, hair, horns, nails, and feather. Due to their unique characteristics of outstanding biocompatibility, great biodegradability, and hydrophilic, keratins have been widely applicated in different fields. In our study, the feather keratin-montmorillonite nanocomposite hydrogels that consist of keratin hydrogels serving as the scaffold support to accommodate endogenous stem cells and montmorillonite is synthesized. The introduction of montmorillonite greatly improves the osteogenic effect of the keratin hydrogels via bone morphogenetic protein 2 (BMP-2)/phosphorylated small mothers against decapentaplegic homolog 1/5/8 (p-SMAD 1/5/8)/runt-related transcription factor 2 (RUNX2) expression. Moreover, the incorporation of montmorillonite into hydrogels can improve the mechanical properties and bioactivity of the hydrogels. The morphology of feather keratin-montmorillonite nanocomposite hydrogels was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of montmorillonite into the keratin hydrogels was confirmed by the energy dispersive spectrum (EDS). We prove that the feather keratin-montmorillonite nanocomposite hydrogels enhance the osteogenic differentiation of BMSCs. Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that feather keratin-montmorillonite nanocomposite hydrogels dramatically stimulated bone regeneration in vivo. Collectively, feather keratin-montmorillonite nanocomposite hydrogels can regulate BMP/SMAD signaling pathway to stimulate osteogenic differentiation of endogenous stem cells and promote bone defect healing, indicating their promising candidate in bone tissue engineering.