Development of Silk Fibroin-Based Non-Crosslinking Thermosensitive Bioinks for 3D Bioprinting.

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 ß-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.

The effects of proteins released from silk mat layers on macrophages.

BACKGROUND: The objective of this study was to evaluate the changes in gene expression after incubation of cells with proteins released from different silk mat layers. METHODS: A silk cocoon from Bombyx mori was separated into four layers of equal thickness. The layers were numbered from 1 to 4 (from the inner to the outer layer). The proteins were released by sonication of a silk mat layer in normal saline. The concentration of proteins was determined by spectrophotometry. They were incubated with RAW264.7 cells, and changes in the expression of genes were evaluated by cDNA microarray analysis and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: Layer 1 and 4 groups had higher protein concentrations compared to those in layer 2 and 3 groups. The genes associated with inflammation and angiogenesis showed significantly higher expression in layer 1 and 4 groups. The results of qRT-PCR were in agreement with those of the cDNA microarray analysis. CONCLUSIONS: The silk mat from the middle portion of the silkworm cocoon yielded a lower protein release and caused an insignificant change in the expression of genes that are associated with inflammation and angiogenesis.

Promoter polymorphism (-590, T/C) of interleukin 4 (IL4) gene is associated with rheumatoid arthritis: An updated meta-analysis.

Rheumatoid arthritis (RA) is a chronic disease. It causes chronic inflammation of the joint. Recent studies suggested that interleukin 4 (IL4) contributes to susceptibility and severity of rheumatoid arthritis (RA). Especially, it was reported that promoter polymorphism (-590, T/C) of IL4 gene has been associated with susceptibility of RA. The aim of present study was to investigate whether the promoter polymorphism (-590, T/C) of IL4 gene is associated with the susceptibility of RA using meta-analysis. And in order to perform meta-analysis, comprehensive meta analysis program was used. Genetic models (co-dominant, dominant, recessive, and allele) were used to determine odds ratios (ORs), 95% confidence intervals (CIs), and P values. Nine case-control studies with case and control design were included in this meta-analysis. Overall, meta-analysis revealed a strong association with susceptibility of RA [OR = 1.303, 95% CI = 1.093-1.554, P = 0.003 in allele model (C vs. T); OR = 1.247, 95% CI = 1.054-1.474, P = 0.010 in dominant model (CC vs. CT + TT); OR = 2.148, 95% CI = 1.263-3.651, P = 0.005 in recessive model (CC + CT vs. TT)]. Our data demonstrated that promoter polymorphism (-590, T/C) of IL4 gene may be contributed to susceptibility of RA. However, more studies with a larger sample size are needed to provide more precise evidence.

Functional recovery guided by an electrospun silk fibroin conduit after sciatic nerve injury in rats.

The aim of this study was to evaluate the regenerative capacity of a newly developed nerve guidance conduit using electrospun silk fibroin (SFNC) implanted in a 10-mm defect of the sciatic nerve in rats. After evaluating the physical properties and cytocompatibility of SFNC in vitro, rats were randomly allocated into three groups: defect only, autograft and SFNC. To compare motor function and abnormal sensation among groups, ankle stance angle (ASA) and severity of autotomy were observed for 10 weeks after injury. Immunostaining with axonal neurofilament (NF) and myelin basic protein (MBP) antibodies were performed to investigate regenerated nerve fibres inside SFNC. ASA increased significantly in the SFNC group at 1, 7 and 10 weeks after injury compared to the defect only group (p<0.05). At one week, mean ASA of the SFNC group was significantly higher than that of the autograft group (p<0.05). Onset and severity of autotomy decreased significantly in the SFNC group compared to other groups (p<0.05). Autotomy in the SFNC group started at 4 weeks and maximally reached toe level. However, the defect only and autograft groups first showed autotomy at 2 and 1 weeks following injury, respectively, and then reached the sole level. Well myelinated nerve fibres stained with NF and MBP were found inside SFNC. In conclusion, SFNC could be helpful in restoring motor function and preventing abnormal sensations after nerve injury.

Powdered Wound Dressing Materials Made from wild Silkworm Antheraea pernyi Silk Fibroin on Full-skin Thickness Burn Wounds on Rats.

PURPOSE: This study evaluated powdered burn wound dressing materials from wild silkworm fibroin in an animal model. METHODS: Fifteen rats were used in this experiment. Full-thickness 2×2 cm burn wounds were created on the back of rats under anesthesia. In the two experimental groups, the wounds were treated with two different dressing materials made from silkworm fibroin. In the Control Group, natural healing without any dressing material was set as control. The wound surface area was measured at five days, seven days and 14 days. Wound healing was evaluated by histologic analysis. RESULTS: By gross observation, there were no infections or severe inflammations through 14 days post-injury. The differences among groups were statistically significant at seven days and 14 days, postoperatively (P <0.037 and 0.001, respectively). By post hoc test, the defect size was significantly smaller in experimental Group 1 compared with the Control Group and experimental Group 2 at seven days postoperatively (P =0.022 and 0.029, respectively). The difference between Group 1 and Group 2 was statistically significant at 14 days postoperatively (P <0.001). Group 1 and control also differed significantly (P =0.002). Group 1 showed a smaller residual scar than the Control Group and Group 2 at 14 days post-injury. Histologic analysis showed more re-epithelization in Groups 1 and 2 than in the Control Groups. CONCLUSION: Burn wound healing was accelerated with silk fibroin spun by wild silkworm Antheraea pernyi. There was no atypical inflammation with silk dressing materials. In conclusion, silk dressing materials can be used for treatment of burn wound.

Soft Tissue Augmentation with Silk Composite Graft.

PURPOSE: The objective of this study was to evaluate the interaction between 4-hexylresorcinol (4HR) and antibody as that affects the performance of a silk-4HR combination graft for soft tissue augmentation in an animal model. METHODS: The silk graft materials consisted of four types: silk+10% tricalcium phosphate (TCP) (ST0), silk+10% TCP+1% 4HR (ST1), silk+10% TCP+3% 4HR (ST3), and silk+10% TCP+6% 4-HR (ST6). The antibody binding assay tested the 4HR effect and scanning electron microscopic (SEM) exam was done for silk grafts. The animal experiment used a subcutaneous pocket mouse model. The graft - SH0 or SH1 or SH3 or SH6 - was placed in a subcutaneous pocket. The animals were killed at one, two, and four weeks, postoperatively. The specimens were subjected to histological analysis and lysozyme assay. RESULTS: Groups with 4HR applied showed lower antibody binding affinity to antigen compared to groups without 4HR. In the SEM examination, there was no significant difference among groups. Histological examinations revealed many foreign body giant cells in ST0 and ST1 group at four weeks postoperatively. Both ST3 and ST6 groups developed significantly lower levels of giant cell values compared to ST0 and ST1 groups (P <0.001) at four weeks postoperatively. In the lysozyme assay, the ST1 and ST3 groups showed denser signals than the other groups. CONCLUSION: 4HR combined silk implants resulted in high levels of vascular and connective tissue regeneration.

Fabrication and evaluation of poly(epsilon-caprolactone)/silk fibroin blend nanofibrous scaffold.

In this study we investigated the blend electrospinning of poly(ϵ-caprolactone) (PCL) and silk fibroin (SF) to improve the biodegradability and biocompatibility of PCL-based nanofibrous scaffolds. Optimal conditions to fabricate PCL/SF (50/50) blend nanofiber were established for electrospinning using formic acid as a cosolvent and three-dimensional (3D) PCL/SF blend nanofibrous scaffolds were prepared by a modified electrospinning process using methanol coagulation bath. The physical properties of 2D PCL/SF blend nanofiber mats and 3D highly porous blend nanofibrous scaffolds were measured and compared. To evaluate cytocompatibility of the 3D blend scaffolds as compared to 3D PCL nanofibrous scaffold, normal human dermal fibroblasts were cultured. It is concluded that biodegradability and cytocompatibility could be improved for the 3D highly porous PCL/SF (50/50) blend nanofibrous scaffold prepared by blending PCL with SF in electrospinning. In addition to the blending of PCL and SF, the 3D structure and high porosity of electrospun nanofiber assemblies may also be important factors for enhancing the performance of scaffolds.

Transduced PEP-1-AMPK inhibits the LPS-induced expression of COX-2 and iNOS in Raw264.7 cells.

AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that plays a central role in cellular metabolic stress. Modulation of nitric oxide (NO) and cyclooxygenase-2 (COX-2) is considered a promising approach for the treatment of inflammation and neuronal diseases. In this study, the AMPK gene was fused in-frame with PEP-1 peptide in a bacterial expression vector to produce a PEP-1-AMPK fusion protein. Expressed and purified PEP-1-AMPK fusion proteins were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose-dependent manner. Furthermore, transduced PEP-1-AMPK fusion protein markedly inhibited LPS-induced iNOS and COX-2 expression. These results suggest that the PEP-1-AMPK fusion protein can be used for the protein therapy of COX-2 and NO-related disorders such as inflammation and neuronal diseases. [BMB reports 2010; 43(1): 40-45].

Low molecular weight silk fibroin increases alkaline phosphatase and type I collagen expression in MG63 cells.

Silk fibroin, produced by the silkworm Bombyx mori, has been widely studied as a scaffold in tissue engineering. Although it has been shown to be slowly biodegradable, cellular responses to degraded silk fibroin fragments are largely unknown. In this study, silk fibroin was added to MG-63 cell cultures, and changes in gene expression in the MG-63 cells were screened by DNA microarray analysis. Genes showing a significant (2-fold) change were selected and their expression changes confirmed by quantitative RT-PCR and western blotting. DNA microarray results showed that alkaline phosphatase (ALP), collagen type-I alpha-1, fibronectin, and transforming growth factor-beta1 expressions significantly increased. The effect of degraded silk fibroin on osteoblastogenic gene expression was confirmed by observing up-regulation of ALP activity in MG-63 cells. The finding that small fragments of silk fibroin are able to increase the expression of osteoblastogenic genes suggests that controlled degradation of silk fibroin might accelerate new bone formation. [BMB reports 2010; 43(1): 52-56].

A combination graft of low-molecular-weight silk fibroin with Choukroun platelet-rich fibrin for rabbit calvarial defect.

OBJECTIVE: The objective of this study was to determine the capabilities of silk fibroin as a biomaterial template for bone formation when mixed with Choukroun platelet-rich fibrin (PRF) in vivo. STUDY DESIGN: Ten New Zealand white rabbits were used for this study and bilateral round shaped defects were formed in the parietal bone (diameter 9.0 mm). The silk fibroin was digested by acid and made into powder (molecular weight <1.0 kDa). The right side (experimental group) received the silk fibroin plus platelet-rich fibroin and the left side (control group) did not receive a graft. Animals were killed at 6 weeks and 12 weeks. The specimens were examined by microscopic computerized tomography (micro-CT). Subsequently, they underwent decalcification and were stained for histologic analysis. RESULTS: There was no significant difference between groups at 6 weeks after operation. In the micro-CT results, however, tissue mineral content in the experimental group at 12 weeks after operation was 132.09 +/- 4.41 and that in the control group was 126.42 +/- 6.62 (P = .011). Tissue mineral density in the experimental group was 2,088.88 +/- 648.34, and that in the control group was 2,029.72 +/- 668.22 (P = .013). The results of the histomorphometric analysis were in accordance with the micro-CT results. The total new bone was 49.86 +/- 7.49% in the control group at 12 weeks after the operation and 59.83 +/- 10.92% in the experimental group (P = .021). CONCLUSION: A combined application of Choukroun PRF with acid-digested silk fibroin showed more rapid bone healing than unfilled control.

Preparation of semi-interpenetrating polymer networks composed of silk fibroin and poloxamer macromer.

A system was designed to utilize silk fibroin (SF) as a matrix for wound dressing. For this system, we prepared a sponge type of porous semi-interpenetrating networks (SIPNs) hydrogel composed of SF and poloxamer 407 macromer to enhance the mechanical and functional properties of SF. The thermal and mechanical properties of the hydrogels as well as their swelling behaviors were studied by means of differential scanning calorimetry, compressive modulus measurement, and gravimetric method, respectively. The morphology and crystalline structure of these SIPN hydrogels were also investigated by scanning electron microscopy (SEM) and wide-angle diffractometry, respectively. Conformational change of SF from random coil to beta-sheet structure was accelerated by formation of SIPNs with poloxamer. The melting temperature of poloxamer in the SIPNs decreased due to the prevention of crystallization by the incorporation of SF. The mechanical strength of SIPNs hydrogel was much higher than those of SF itself or SF/poloxamer blend and increased with the poloxamer content. The equilibrium water content of SF was remarkably increased by formation of SIPNs with poloxamer due to the hydrophilicity of poloxamer. The crystallinity and morphology of SIPNs hydrogel were affected by SIPNs hydrogel composition.

The role of formic acid in solution stability and crystallization of silk protein polymer.

In this paper, the regenerated silk fibroin (SF) solution dissolved in formic acid was used as a model protein to understand the role of formic acid in solution stability and crystallization of protein-based materials. The molecular decomposition of SF did not occur for the dissolution process in formic acid within 1-2 days of storage times. The beta-sheet crystallization of SF molecules was occurred by the elimination of formic acid upon drying. The SF molecules in formic acid solution are stable and have low hydrodynamic radius values. This may be closely related to the fact that formic acid has two opposite functions of dissolution and crystallization simultaneously. The turbidity, dynamic light scattering and FTIR measurements elucidate that the solution stability and crystallization of SF are attributed to compact molecular shape of SF in formic acid, resulted from the molecular interactions between formic acid and polar groups in SF molecules.

Preparation of self-assembled silk sericin nanoparticles.

Silk sericin (SS) possessing moisture-retaining property was reacted with activated poly(ethylene glycol) (PEG) to obtain self-assembled SS nanoparticles. The aliphatic and aromatic hydroxyl groups of serine and tyrosine residues as the reaction sites in SS were clarified by amino acid analysis and 1H NMR spectroscopy, respectively. From IR and circular dichroism (CD) measurements, introduction of PEG into SS induced the conformational change from random coil to beta-sheet. DSC thermogram of sericin-PEG conjugate suggests that mutual miscibility between PEG and SS chains was poor. Nanoparticles of sericin-PEG conjugate with sizes measured by dynamic light scattering ranging about 200-400 nm in diameter, were prepared by the diafiltration method. Shape of sericin-PEG conjugate nanoparticles observed by scanning and transmission electron microscopes was spherical. The results suggest that sericin-PEG conjugates are self-associated to form spherical nanoparticles through hydrophobic interaction.