The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group (p = 0.044) and the AL group (p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering.

Bone morphogenic protein-2/4 (BMP-2/4) is an osteoinductive protein that accelerates osteogenesis when administered to bony defects. Sericin is produced by silkworms, and has a biological activity that differs depending on the degumming method used. Our results indicated that the high molecular weight fraction of silk sericin (MW > 30 kDa) obtained via sonication had a more abundant β-sheet structure than the low molecular weight fraction. Administration of the β-sheet structure silk sericin increased BMP-2/4 expression in a dose-dependent manner in RAW264.7 cells and human monocytes. This sericin increased the expression levels of toll-like receptor (TLR)-2, TLR-3, and TLR-4 in RAW264.7 cells. Application of a TLR-2 antibody or TLR pathway blocker decreased BMP-2/4 expression following sericin administration. In the animal model, the bone volume and BMP-2/4 expression were higher in rats treated with a sericin-incorporated gelatin sponge than in rats treated with a gelatin sponge alone or a sponge-incorporated with denatured sericin. In conclusion, sericin with a more abundant β-sheet structure increased BMP-2/4 expression and bone formation better than sericin with a less abundant β-sheet structure.

Sericin is a 10-to-400 kDa hydrophilic protein with high serine content and is a silk constituent together with fibroin. It is produced in the middle silk gland of the silkworm and encoded by four sericin genes. The molecular weight of sericin and its biological activity vary depending on the extraction method employed. Its chemical structure, in terms of random coil and β-sheet conformations, also differs with the extraction method, thereby extending its applications in various fields. Sericin, which was discarded in the textile industry in the past, is being applied and developed in the biomedical field, owing to its biological properties. In particular, many studies are underway in the field of tissue engineering, evaluating its applicability in burn dressing, drug delivery, bone regeneration, cartilage regeneration, and nerve regeneration.

Silk fibroin (SF)-based materials are exposed to both natural and artificial ultraviolet (UV) light during preparation or administration. However, the effects of UV irradiation on SF films prepared under different conditions have not yet been described in detail. In this study, four SF films with different molecular weight (MW) distribution were fabricated using SF solutions, which were prepared by dissolving degummed SF for 0.5–24 h. We observed UV (365 nm) irradiation on SF films induced the increase of yellowness and absorbance at 310 nm of SF films, indicating the formation of new photo-products and di-tyrosine bonds by photo-oxidation. Due to di-tyrosine cross-links between SF chains, UV-irradiated SF films were not fully dissociated in urea solution. In addition to formation of new products, UV reduced the crystallinity of SF films by breaking hydrogen bonds of β-sheet conformation. Unlike the UV-induced decomposition of physical interactions, UV did not affect the covalent bonds (i.e., peptide bonds). Through these experiments, we could expect that SF with higher MW was more susceptible and SF with lower MW was more resistant to UV-induced photo-oxidation and photo-degradation. These results provide useful information about UV-induced aging of SF-based materials under natural sunlight and UV irradiating conditions.

Three-dimensional (3D) bioprinting holds great promise for tissue engineering, allowing cells to thrive in a 3D environment. However, the applicability of natural polymers such as silk fibroin (SF) in 3D bioprinting faces hurdles due to limited mechanical strength and printability. SF, derived from the silkworm Bombyx mori, is emerging as a potential bioink due to its inherent physical gelling properties. However, research on inducing thermosensitive behavior in SF-based bioinks and tailoring their mechanical properties to specific tissue requirements is notably lacking. This study addresses these gaps through the development of silk fibroin-based thermosensitive bioinks (SF-TPBs). Precise modulation of gelation time and mechanical robustness is achieved by manipulating glycerol content without recourse to cross-linkers. Chemical analysis confirms β-sheet conformation in SF-TPBs independent of glycerol concentration. Increased glycerol content improves gelation kinetics and results in rheological properties suitable for 3D printing. Overall, SF-TPBs offer promising prospects for realizing the potential of 3D bioprinting using natural polymers.

Histone deacetylases (HDACs) are key enzymes for post-translational modification and influence on various cellular activities. Thus, HDACs are associated with many diseases and their inhibitors have clinical significance. Here, 4-Hexylresorcinol (4HR) was studied as an inhibitor for class I HDACs using the HDAC inhibitor (HDACi) Trichostatin-A as a positive control. The 4HR was administered 1–100 μM to human umbilical endothelial cells (HUVECs) and the HDAC expression and activity were examined. The 4HR decreased the expression level of HDAC1, 3, 4, and 5 in a time and dose-dependent manner. The 4HR also increased acetylated lysine and decreased HDAC activity significantly (p < 0.05). Collectively, 4HR was a new class I HDAC inhibitor that reduced the expression and activity of HDAC in HUVECs.