Fructose-mineralized black phosphorus for syncretic bone regeneration and tumor suppression.

Black phosphorus (BPs) nanosheets with their inherent and selective chemotherapeutic effects have recently been identified as promising cancer therapeutic agents, but challenges in surface functionalization hinder satisfactory enhancement of their selectivity between tumors and normal cells. To address this issue, we developed a novel biomineralization-inspired strategy to synthesize CaBPs-Na2FDP@CaCl2 nanosheets, aiming to achieve enhanced and selective anticancer bioactivity along with accelerated osteoblast activity. Benefiting from the in situ mineralization and fructose modification, CaBPs-Na2FDP@CaCl2 exhibited improved pH-responsive degradation behavior and targeted therapy for osteosarcoma. The in vitro results indicated that CaBPs-Na2FDP@CaCl2 exhibited efficient uptake and quick degradation by GLUT5-positive 143B osteosarcoma cells, enhancing BPs-driven chemotherapeutic effects through ATP level disturbance-mediated apoptosis of tumor cells. Moreover, CaBPs-Na2FDP@CaCl2 underwent gradual degradation into PO43-, Ca2+ and fructose in MC3T3-E1 cells, eliminating systemic toxicity. Intracellular Ca2+ bound to calmodulin (CaM), activating Ca2+/CaM-dependent signaling cascades, thereby enhancing osteoblast differentiation and mineralization in pro-osteoblastic cells. In vivo experiments affirmed the anti-tumor capability, inhibition of tumor recurrence and bone repair promotion of CaBPs-Na2FDP@CaCl2. This study not only broadens the application of BPs in bone tumor therapy but also provides a versatile surface functionalization strategy for nanotherapeutic agents.

Effects of matrix viscoelasticity on cell-matrix interaction, actin cytoskeleton organization, and apoptosis of osteosarcoma MG-63 cells.

Many recent reports have shown the effects of viscoelasticity of the extracellular matrix on the spreading, migration, proliferation, survival and cell-matrix interaction of mesenchymal stem cells and normal cells. However, the effect of matrix viscoelasticity on the behavior of tumor cells is still in the state of preliminary exploration. To this aim, we prepared a viscoelastic hydrogel matrix with a storage modulus of about 2 kPa and a loss modulus adjustable from 0 to 600 Pa, through adding linear alginate and regulating the compactness of a polyacrylamide covalent network. Overall, the addition of viscous components inhibited the apoptosis of osteosarcoma MG-63 cells, while it promoted their spreading and proliferation and in particular led to a well-developed cytoskeleton organization. However, with the increase of the viscous fraction, this trend was reversed, and the apoptosis of MG-63 cells gradually increased with gradually decreased spreading and proliferation, accompanied by a surprising manner change of the cytoskeleton from fusiform cells dominated by focal adhesion to dendritic cells dominated by pseudopodia. Besides the upregulation of MAPK, Ras, Rap1 and PI3K-Akt pathways commonly involved in mechanotransduction, the upregulation of the Wnt pathway and inhibited endoplasmic reticulum stress-mediated apoptosis were observed for the viscous matrix with a low loss modulus. The high viscosity matrix showed additional involvement of Hippo and NF-kappa B signaling pathways related to the cell-matrix interaction, with downregulation of the endoplasmic reticulum stress pathway and upregulation related to mitochondrial organization. Our study would provide insight into the effect of viscosity on fundamental behaviors of tumor cells and might have important implications in designing antitumor materials.

Matrix Stiffness Regulated Endoplasmic Reticulum Stress-mediated Apoptosis of Osteosarcoma Cell through Ras Signal Cascades.

The modulating effects of matrix stiffness on spreading and apoptosis of tumor cells have been well recognized. Nevertheless, the detail road map leading to the apoptosis and the underlying mechanisms governing the cell apoptosis have remained to be elucidated. To this aim, we provided a tunable elastic hydrogel matrix that promoted cell adhesion by modifying the surface of polyacrylamide with polydopamine, with stiffness value of 1, 10, 30, and 250 kPa, respectively. While the cell spreading increased and the apoptosis decreased with the matrix stiffness, such modulating effect of matrix on cell spreading exhibited different time evolvement behaviors as a function of stiffness, which likely led to surprisingly similar apoptosis rates for the 30 kPa and 250 kPa samples. Matrix stiffness mediated the spreading and apoptosis of MG-63 cells by regulating cell adhesion to matrix and in particular cytoskeletal organization, which was dependent on Ras, Rap1 and PI3K-Akt signaling pathways and finally led to the apoptosis of cancer cells dominated by endoplasmic reticulum stress pathway. Our results provided an insight into the regulation of tumor cell fate by the mechanical clues of ECM, which would have implication for future cancer research and the design of novel anticancer materials.

Magnetic chitosan hydrogel induces neuronal differentiation of neural stem cells by activating RAS-dependent signal cascade.

Our aim was to modulate magnetic cues to influence the differentiation of neural stem cell (NSC) into neuron during nerve repair and to explore corresponding mechanisms. Here, a magnetic hydrogel composed of chitosan matrices and magnetic nanoparticles (MNPs) with different content was prepared as the magnetic-stimulation platform to apply intrinsically-present magnetic cue and externally-applied magnetic field to NSC grown on the hydrogel. The MNP content had regulatory effects on neuronal differentiation and the MNPs-50 samples exhibited the best neuronal potential and appropriate biocompatibility in vitro, as well as accelerated the subsequent neuronal regeneration in vivo. Remarkably, the use of proteomics analysis parsed the underlying mechanism of magnetic cue-mediated neuronal differentiation form the perspective of protein corona and intracellular signal transduction. The intrinsically-present magnetic cues in hydrogel contributed to the activation of intracellular RAS-dependent signal cascades, thus facilitating neuronal differentiation. Magnetic cue-dependent changes in NSCs benefited from the upregulation of adsorbed proteins related to "neuronal differentiation", "cell-cell interaction", "receptor", "protein activation cascade", and "protein kinase activity" in the protein corona. Additionally, magnetic hydrogel acted cooperatively with the exterior magnetic field, showing further improving neurogenesis. The findings clarified the mechanism for magnetic cue-mediated neuronal differentiation, coupling protein corona and intracellular signal transduction.

Stiff and Tough Hydrogels Prepared Through Integration of Ionic Cross-linking and Enzymatic Mineralization.

Enzymatic mineralization has become an effective approach to enhancing the stiffness of hydrogels for bone tissue engineering, but generally with limited toughness. On the other hand, double network cross-linking provides hydrogel with enhanced toughness. In this study, we integrated double cross-linking method with enzymatic mineralization to synthesize stiff and tough hydrogels. We have synthesized three kinds of sodium alginate-polyacrylamide (SA-PAM) double-network hydrogels and systematically compared the composition and structure differences, mechanical properties, and biological properties of the different hydrogels in the absence and presence of mineralization. In particular, we examined the role of specific cross-linking ions, i.e., calcium, zinc and strontium ions, in modulating the mineralization process. Synergistic effect of ionic cross-linking and enzymatic mineralization was clearly observed with dramatic increase in compressive modulus. In particular, mineralized hydrogel cross-linked with Sr2+ showed the highest compressive Young's modulus of 17.28 ± 3.56 MPa, which was 37 times of that of the original hydrogel. In addition, it had the highest tensile Young's modulus at 2.60 ± 0.25 MPa and 84 ± 5.5% elongation at break. Such synergistic effect from Sr2+ was attributed to a more uniformed mineralization process due to the early initiation of a more homogeneous nucleation process and subsequent denser mineralized structure. Cellular study also suggested that cell proliferation, adhesion and osteogenic differentiation were improved as a result of enzymatic mineralization. Our results provided an effective way for the preparation of stiff and tough hydrogels with osteogenesis, and demonstrated potential in bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: Hydrogels with excellent stiffness, stability and biocompatibility have attracted significant attentions in the bone tissue engineering applications. Our results suggested that the synergistic effect of ionic cross-linking and enzymatic mineralization rendered more enhancement of the compressive and tensile stiffness of SA-PAM DN hydrogels, as well as the toughness, swelling stability and cellular response. In particular, mineralized hydrogel cross-linked with Sr2+ showed the highest compressive Young's modulus of 17.28 ± 3.56MPa, which was 37 times of that of the original hydrogel. Such synergistic effect from Sr2+ was attributed to a more uniformed mineralization process. The cell proliferation, adhesion and osteogenic differentiation were greatly improved as a result of enzymatic mineralization, where the MSCs cultured on strontium ion cross-linked mineralized hydrogel showed the best performance.