Polyphenolic Carbon Quantum Dots with Intrinsic Reactive Oxygen Species Amplification for Two-Photon Bioimaging and In Vivo Tumor Therapy.

Recent studies indicate that mitochondrial dysfunctions and DNA damage have a critical influence on cell survival, which is considered one of the therapeutic targets for cancer therapy. In this study, we demonstrated a comparative study of the effect of polyphenolic carbon quantum dots (CQDs) on in vitro and in vivo antitumor efficacy. Dual emissive (green and yellow) shape specific polyphenolic CQDs (G-CQDs and Y-CQDs) were synthesized from easily available nontoxic precursors (phloroglucinol), and the antitumor property of the as-synthesized probe was investigated as compared to round-shaped blue emissive CQDs (B-CQDs) derived from well-reported precursor citric acid and urea. The B-CQDs had a nuclei-targeting property, and G-CQDs and Y-CQDs had mitochondria-targeting properties. We have found that the polyphenol containing CQDs (at a dose of 100 µg mL-1) specifically attack mitochondria by excess accumulation, altering the metabolism, inhibiting branching pattern, imbalanced Bax/Bcl-2 homeostasis, and ultimately generating oxidative stress levels, leading to oxidative stress-induced cell death in cancer cells in vitro. We show that G-CQDs are the main cause of oxidative stress in cancer cells because of their ability to produce sufficient •OH- and 1O2 radicals, evidenced by electron paramagnetic resonance spectroscopy and a terephthalic acid test. Moreover, the near-infrared absorption properties of the CQDs were exhibited in two-photon (TP) emission, which was utilized for TP cellular imaging of cancer cells without photobleaching. The in vivo antitumor test further discloses that intratumoral injection of G-CQDs can significantly augment the treatment efficacy of subcutaneous tumors without any adverse effects on BalB/c nude mice. We believe that shape-specific polyphenolic CQD-based nanotheranostic agents have a potential role in tumor therapy, thus proving an insight on treatment of malignant cancers.

A 3D Bioprinted Nanoengineered Hydrogel with Photoactivated Drug Delivery for Tumor Apoptosis and Simultaneous Bone Regeneration via Macrophage Immunomodulation.

One of the significant challenges in bone tissue engineering (BTE) is the healing of traumatic tissue defects owing to the recruitment of local infection and delayed angiogenesis. Herein, a 3D printable multi-functional hydrogel composing polyphenolic carbon quantum dots (CQDs, 100 µg mL-1 ) and gelatin methacryloyl (GelMA, 12 wt%) is reported for robust angiogenesis, bone regeneration and anti-tumor therapy. The CQDs are synthesized from a plant-inspired bioactive molecule, 1, 3, 5-trihydroxybenzene. The 3D printed GelMA-CQDs hydrogels display typical shear-thinning behavior with excellent printability. The fabricated hydrogel displayed M2 polarization of macrophage (Raw 264.7) cells via enhancing anti-inflammatory genes (e.g., IL-4 and IL10), and induced angiogenesis and osteogenesis of human bone mesenchymal stem cells (hBMSCs). The bioprinted hBMSCs are able to produce vessel-like structures after 14 d of incubation. Furthermore, the 3D printed hydrogel scaffolds also show remarkable near infra-red (NIR) responsive properties under 808 nm NIR light (1.0 W cm-2 ) irradiation with controlled release of antitumor drugs (≈49%) at pH 6.5, and thereby killing the osteosarcoma cells. Therefore, it is anticipated that the tissue regeneration and healing ability with therapeutic potential of the GelMA-CQDs scaffolds may provide a promising alternative for traumatic tissue regeneration via augmenting angiogenesis and accelerated immunomodulation.

Polyphenol derived bioactive carbon quantum dot-incorporated multifunctional hydrogels as an oxidative stress attenuator for antiaging and in vivo wound-healing applications.

Upregulation of certain enzymes, such as collagenase, tyrosinase, and elastase, is triggered by several extrinsic environmental factors, such as temperature, UV radiation, humidity, and stress, and leads to elasticity loss and skin pigmentation. Herein, dual-emissive polyaromatic carbon quantum dots (CQDs) with abundant phenolic moieties, that is green and yellow CQDs (G-CQDs and Y-CQDs, respectively), were prepared using a three-fold symmetric molecule, 1,3,5-trihydroxybenzene. The significant inhibition efficacy of the fabricated CQDs against collagenase, elastase, and tyrosinase, which play important roles in skin aging, revealed their excellent antiaging potential. Y-CQDs with large polyphenolic-polyaromatic domains and abundant -OH groups exhibited high enzyme inhibitory efficacy against skin aging, and their collagenase, elastase, and tyrosinase inhibitory efficacies were ∼75 ± 4.2%, ∼52 ± 3.1%, and ∼35.3 ± 4.2%, respectively, at a concentration of 100 µg mL-1. The most critical factor that delays wound healing is oxidative stress, which is caused by the overproduction of free radicals around inflamed tissue. CQDs were effective in suppressing UV-induced reactive oxygen species at the cellular level and improved the cell viability. Subsequently, CQD-incorporated dual-emissive biocompatible gelatin-methacryloyl hydrogels were constructed as wound dressing materials to promote wound healing via inducing the proliferation of fibroblasts, enhancing cell migration and alleviating inflammation and to provide antiaging benefits. Our results demonstrated that the fabricated CQDs with remarkable optical features, low cytotoxicity, and excellent antioxidant and antiaging properties can be used as bio-imaging probes, antiaging agents, and wound dressing materials for oxidative stress-related diseases in the nanomedicine and cosmetics industries.

Functionalized chitosan/spherical nanocellulose-based hydrogel with superior antibacterial efficiency for wound healing.

Cellulose nanomaterials have received significant interest due to their superior physicochemical properties and biocompatibility. The nanomaterials-based hydrogel patches are widely explored for skin regeneration. However, the injectability and adhesiveness of the hydrogels are crucial challenges for tissue engineering applications. To overcome these, we synthesized an injectable and adhesive hydrogel of spherical nanocellulose (s-NC) reinforced carboxymethyl chitosan for rapid skin regeneration. The s-NC exhibited improved cellular activity than cellulose nanocrystals. The hydrogels exhibited adhesive and injectability potentials and were molded in the desired configurations. An enhanced conductivity was observed in s-NC added hydrogels than the pure polymer hydrogel. The skin regeneration potential of the hydrogel scaffolds was also examined in the rats using the wound healing model. The composite scaffolds also showed improved antibacterial potential. Taken together, the developed hydrogels have the potential and can be explored as a promising biomaterial for enhanced skin regeneration applications.

Two-photon excitable membrane targeting polyphenolic carbon dots for long-term imaging and pH-responsive chemotherapeutic drug delivery for synergistic tumor therapy.

Long-term dynamic tracking of cells with theranostic properties remains challenging due to the difficulty in preparing and delivering drugs by probes. Herein, we developed highly fluorescent one- and two-photon (OP and TP) excitable polyphenolic carbon quantum dots (CQDs) for excellent membrane-targeting and drug delivery properties for synergistic tumor therapy. The green-emissive CQDs (g-CQDs) were synthesized from a three-fold symmetric polyphenolic molecule, phloroglucinol (C3h; symmetry elements: E, C3, C32, σh, S3, and S3-1), in a sulfuric acid medium. Doxorubicin (Dox) was loaded onto the g-CQDs via electrostatic interaction, resulting in a loading efficiency and content of 54.62% and 323.25 µg mL-1, respectively. The g-CQDs@Dox complex exhibited a higher rate of cell killing efficiency at both pH 5.0 and 6.5, with higher reactive oxygen species (ROS) generation due to the greater Dox accumulation in the tumor cells. In addition, TP cell imaging displayed excellent membrane-targeting properties with less photobleaching ability in tumor cells. The in vivo studies confirmed that the g-CQDs@Dox complex has higher affinity towards tumor cells, better inhibitory effects, and an absence of systemic toxicity. Therefore, our developed nanocarrier exhibited better cell imaging, drug delivery, and tumor-targeting properties, and could be used as a "smart" probe for synergistic tumor therapy.

3D-printable chitosan/silk fibroin/cellulose nanoparticle scaffolds for bone regeneration via M2 macrophage polarization.

Biopolymers-induced immune microenvironment exhibited prominent effects on bone regeneration. Osteo-immunomodulatory responses of cellulose nanoparticles incorporated chitosan hydrogel scaffolds have not yet been reported. The objective of this study was to monitor the synergistic effects of silk fibroin and cellulose nanoparticles on the immune-modulatory behavior of chitosan biopolymer scaffolds. 3D-printed biodegradable cellulose nanoparticles-reinforced chitosan/silk fibroin (CS/SF/CNPs) scaffolds were fabricated and characterized by different spectroscopic techniques. The improved rheological and recovery strength was observed in CS/SF/CNPs hydrogels than pure polymer hydrogels. A significant shift from M1 â†’ M2 macrophages polarization occurred in the CS/SF/CNPs scaffolds treated groups than the control after 3 days of incubation, showing its immune-modulatory potential. Osteo-immunomodulatory effects of the fabricated scaffolds were analyzed on human bone marrow-derived mesenchymal stem cells (hBMSCs), with macrophages-derived conditioned media (M-CM). Enhanced bone regeneration was observed in the calvaria defect rat model, indicating that the fabricated scaffolds are promising materials for bone-healing applications.

3D-printed bioactive and biodegradable hydrogel scaffolds of alginate/gelatin/cellulose nanocrystals for tissue engineering.

The 3D-printed hybrid biodegradable hydrogels composed of alginate, gelatin, and cellulose nanocrystals (CNCs) were prepared to provide a favorable environment for cell proliferation, adhesion, nutrients exchange, and matrix mineralization for bone tissue engineering (BTE) applications. The hybrid scaffolds exhibited enhanced mechanical strength compared to the pure polymer scaffolds. The biocompatibility, differentiation potential, and bone regeneration potential of the printed scaffolds were evaluated by DAPI staining, live-dead assay, alizarin Red-S (ARS) staining, real-time PCR (qRT-PCR), and µCT analysis, respectively. Enhanced cell proliferation has occurred 1% CNC/Alg/Gel scaffolds compared to the control. The cells were adequately adhered to the scaffold and exhibited the flattened structure. Improved mineralization was observed in the 1% CNC/Alg/Gel scaffolds' presence than the control, showing their mineralization efficiency. A significant enhancement in the expression of osteogenic-specific gene markers (Runx2, ALP, BMP-2, OCN, OPN, BSP, and COL1) has occurred with 1% CNC/Alg/Gel than the control, indicating their osteogenic potential. Furthermore, enhanced bone formation was observed in the scaffolds treated groups than the control in the calvaria critical-sized defects (CCD-1) model, suggesting their improved bone regeneration potential. Therefore, the fabricated scaffolds have the potential to explore as a biomaterial for tissue engineering.

Bioactive electrospun nanocomposite scaffolds of poly(lactic acid)/cellulose nanocrystals for bone tissue engineering.

Poly(lactic acid) (PLA)/cellulose nanocrystal (CNC) composite scaffolds were fabricated using an electrospinning technique to evaluate the influence of CNCs on the biocompatibility and osteogenic potential of PLA. The scaffolds were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction pattern (XRD), transmission electron microscopy (TEM), and atomic force microscopy (AFM). A significant enhancement of the mechanical properties occurred in the composite scaffolds compared to pure polymer. This is due to the stronger interactions between the polymer chains and CNCs. The composite scaffolds exhibited higher thermal stability compared to pure polymer. Notably, excellent adhesion and proliferation was observed in the presence of the fabricated composite scaffolds, indicating their superior biocompatibility. Higher mineralization was noted on the surface of composite scaffolds. The fabricated scaffolds were significantly covered by the cultured cells and exhibited greater fluorescence intensity vis-à-vis control. Additionally, the fact that higher expression of osteogenic gene markers was observed in composite scaffolds confirms their enhanced osteogenic potential. The bone regeneration potential of the fabricated scaffold was monitored in a rat calvarial defect model after 3 weeks of treatment. The fabricated scaffold demonstrated excellent biocompatibility and superior osteoinductivity. Therefore, the fabricated scaffolds possess potential to be used as a biomaterial for tissue engineering applications.

Evaluation of the Osteogenic Potential of Stem Cells in the Presence of Growth Hormone under Magnetic Field Stimulation.

The human alveolar bone-derived mesenchymal stem cells (hABMSCs) are considered an attractive source for the development of bone tissues. However, their mechanism of action is still unclear. This work aimed to investigate the potential of the natural human growth hormone (NHGH) derived from stem cells under magnetic field (MF) stimulation for tissue engineering by exploring the paracrine or autocrine effects of hABMSCs in vitro. The secretion of anti-inflammatory cytokines and growth factors from hABMSCs was profoundly affected by the intensity of the applied MFs. The effects of stimulated MFs on vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) production were quantified by an ELISA kit. Notably, higher cell metabolic activity was observed in MF stimulation compared to the control, and this was more prominent in 130 mT strength of MF. An enhancement in the production of VEGF and BMP-2 was noted in MFs compared to the control. Moreover, higher accumulation of osteogenesis-related genes has occurred in MFs than the control. Furthermore, a significant enhancement in cell metabolic activity and mineralized nodule formations was spotted in the presence of NHGH via MF stimulation; vis-à-vis, MF stimulation only through autocrine and paracrine effects demonstrated the better osteogenic potential of NHGH in the presence of MFs for tissue engineering applications.

Synergistic effects of orbital shear stress on in vitro growth and osteogenic differentiation of human alveolar bone-derived mesenchymal stem cells.

Cellular behavior is dependent on a variety of physical cues required for normal tissue function. In order to mimic native tissue environments, human alveolar bone-derived mesenchymal stem cells (hABMSCs) were exposed to orbital shear stress (OSS) in a low-speed orbital shaker. The synergistic effects of OSS on proliferation and differentiation of hABMSCs were investigated. In particular, we induced the osteoblastic differentiation of hABMSCs cultured in the absence of OM by exposing hABMSCs to OSS (0.86-1.51 dyne/cm(2)). Activation of Cx43 was associated with exposure of hABMSCs to OSS. The viability of cells stimulated for 10, 30, 60, 120, and 180 min/day increased by approximately 10% compared with that of control. The OSS groups with stimulation of 10, 30, and 60 min/day had more intense mineralized nodules compared with the control group. In quantification of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) protein, VEGF protein levels under stimulation for 10, 60, and 180 min/day and BMP-2 levels under stimulation for 60, 120, and 180 min/day were significantly different compared with those of the control. In conclusion, the results indicated that exposing hABMSCs to OSS enhanced their differentiation and maturation.

Effects of electromagnetic fields on osteogenesis of human alveolar bone-derived mesenchymal stem cells.

This study was performed to investigate the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMFs) on the proliferation and differentiation of human alveolar bone-derived mesenchymal stem cells (hABMSCs). Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined not merely the effect of ELF-PEMFs on cell proliferation, alkaline phosphatase (ALP) activity, and mineralization of the extracellular matrix but vinculin, vimentin, and calmodulin (CaM) expressions in hABMSCs during osteogenic differentiation. Exposure of hABMSCs to ELF-PEMFs increased proliferation by 15% compared to untreated cells at day 5. In addition, exposure to ELF-PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis within 2 weeks. ELF-PEMFs also increased vinculin, vimentin, and CaM expressions, compared to control. In particular, CaM indicated that ELF-PEMFs significantly altered the expression of osteogenesis-related genes. The results indicated that ELF-PEMFs could enhance early cell proliferation in hABMSCs-mediated osteogenesis and accelerate the osteogenesis.

Enhanced osteogenesis of human alveolar bone-derived mesenchymal stem cells for tooth tissue engineering using fluid shear stress in a rocking culture method.

This study instituted a simple approach to stimulate alveolar bone regeneration for tooth tissue engineering by controlling effects of low fluid dynamic shear stress (LFDSS) on growth and differentiation in vitro. Human alveolar bone-derived mesenchymal stem cells (hABMSCs) harvested from human mandibular alveolar bone were cultured with LFDSS to generate cultures containing bone-like formations. To distinguish between osteodifferentiation and bone-like formation, cells were cultured either with or without fluid shear stress. The calcium content and alkaline phosphatase (ALP) activity of hABMSCs were used as indicators of osteogenesis. Cell viability and proliferation after stimulating with LFDSS for 10-60 min/day were higher than with longer stimulations. Mineralized nodules formed when osteoblasts were cultured with an induction medium, a marker of osteogenic differentiation. ALP activity tended to increase after 10 and 60 min/day of stimulation. In addition, LFDSS conditions also increased gene expression of IBSP, RUNX2, COL-I, ALP, OCN, and OPN, as shown by reverse transcriptase-polymerase chain reaction. From the results of a proteomics array, LFDSS groups were intensely expressed with several factors (EGF, HGF, IGF, TGF, and PDGF). Furthermore, CD146 and Stro-1 expression increased in cells treated with 30 min/day and decreased in cells treated with 120 min/day, as determined by cell surface antigen analysis by fluorescence-activated cell-sorting analysis. These results strongly showed that LFDSS at the proper intensity and time enhanced the differentiation and maturation of hABMSCs. In conclusion, an appropriate level of LFDSS can potently and positively modulate proliferation and differentiation in hABMSCs.