In this study, we report for the first time a novel silk fibroin (SF) nanofibrous films with robust mechanical properties that was fabricated by directly dissolving silk in CaCl2-formic acid solution. CaCl2-FA dissolved silk rapidly at room temperature, and more importantly, it disintegrated silk into nanofibrils instead of separate molecules. The morphology of nanofibrils crucially depended on CaCl2 concentrations, which resulted in different aggregation nanostructure in SF films. The SF film after drawing had maximum elastic modulus, ultimate tensile strength, and strain at break reaching 4 GPa, 106 MPa, and 29%, respectively, in dry state and 206 MPa, 28 MPa, and 188%, respectively, in wet state. Moreover, multiple yielding phenomena and substantially strain-hardening behavior was also observed in the stretched films, indicating the important role played by preparation method in regulating the mechanical properties of SF films. These exceptional and unique mechanical properties were suggested to be caused by preserving silk nanofibril during dissolution and stretching to align these nanofibrils. Furthermore, the SF films exhibit excellent biocompatibility, supporting marrow stromal cells adhesion and proliferation. The film preparation was facile, and the resulting SF films manifested enhanced mechanical properties, unique nanofibrous structures, and good biocompability.

The current treatments for severe skin injury all involve skin grafting. However, there is a worldwide shortage of donor skin tissue. In this study, we examined the advantages of using human amniotic fluid stem (hAFS) cells in skin wound healing. In vitro, hAFS cells differentiate into keratinocytes (termed hAFS-K). Like keratinocytes, hAFS-K cells express the markers K5, K14, K10 and involucrin; display typical cellular structure, including a tonofibril-rich cytoplasm; and construct a completely pluristratified epithelium in 3D culture. In vivo, in a mouse excisional wound model, GFP-positive hAFS cells participate in wound repair. Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes. Real-time PCR results confirmed that hAFS cells can initiate and promote early-stage repair of skin damage. During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-β1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1β, IL-6, TNF-α, Cox2 and Mac3. In hAFS cells, the negative co-stimulatory molecule B7H4 regulates low immunogenicity, which can provide a modest inflammatory reaction microenvironment for wound repair. Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.

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The NOD-like receptor protein 3/caspase-1 inflammasome can be activated in human dental pulp tissue and fibroblasts; however, the underlying mechanisms are poorly understood. In the present study, lipopolysaccharide (LPS) was used to treat dental pulp cells to establish an inflammation model. Cell viability was examined by sulforhodamine B assay. Interleukin (IL)-1β, caspase-1, microtubule-associated protein-1 light chain 3-II/I and p62 were determined by western blotting and ELISA. The phosphorylation (p-) levels of NF-κB and NF-κB inhibitor (IκB)α protein were observed by western blotting. The results demonstrated that LPS induced pyroptotic cell death in cultured dental pulp cells, which was supported by the increased levels of IL-1β, IL-18 and caspase-1. Rapamycin and 3-methyladenine (3-MA) were used to activate and inhibit autophagy, and it was observed that LPS increased autophagy and rapamycin reduced LPS-induced dental pulp cell pyroptosis. However, 3-MA aggravated LPS-induced dental pulp cell pyroptosis. In addition, LPS inhibited the expression of IκBα, but increased the expression of p-NF-κB. Compared with the LPS group, 3-MA further inhibited the expression of IκBα but promoted the expression of p-NF-κB. However, rapamycin produced the opposite results to LPS. Under LPS treatment, the NF-κB pathway inhibitor BAY11-7082 further enhanced the inhibitory effects of rapamycin, but inhibited the promoting effects of 3-MA on the protein expression levels of IL-1β and caspase-1. The results of the present study demonstrated that there is an important crosstalk between autophagy, pyroptosis and the NF-κB pathway, and that the modulation of pyroptosis in dental pulp cells may be a promising strategy to pulpitis therapy.

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