Effect of Silver Sulfadiazine and Metallic Ions on Properties of Thai Silk Fibroin/Gelatin Films for Anti-Bacterial Applications

This study aimed to develop Thai silk fibroin/gelatin (SF/GA) films incorporating various concentrations of silver sulfadiazine (SSD) and to investigate the effects of SSD and metallic ions (Ag(I) and Cu(II)) on chemical conformation of the SF/GA films. We found that the incorporation of SSD in the films changed the conformation of silk fibroin protein by increasing β-sheet content. The same phenomena was also observed with the other 2 metallic ions, Ag(I) and Cu(II), incorporated in the SF/GA films. This effect of metallic ions on the SF conformation transition in the SF/GA films may be similar to the phenomenon occurred during natural spinning process of the Bombyx mori silkworm. The SF/GA films incorporating SSD had therefore more stability, less water-insoluble fraction and extended degradation rate than the SF/GA films without SSD due to the higher content of stable β-sheet conformation. When cultured with L929 mouse fibroblast cells according to ISO 10993 part 5 standard, the SF/GA films incorporating SSD at all concentrations showed no cytotoxicity. The SSD released from the films also showed obvious anti-bacterial activity against Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. We suggested that the SF/GA films incorporating SSD may be useful for some medical applications in which anti-bacterial effect was required

Preconditioning Methods in Cartilage Tissue Engineering: Influences of Silk Material Properties and Hypoxia on Chondrogenesis

Cartilage has limited intrinsic healing potential, due to the low cell density and the lack of blood supply. Current treatments for cartilage repair rarely restore full structure and function to the native state. Tissue engineering holds promise to create cartilage grafts capable to withstand the stresses present in joints. More than 90% of articular cartilage tissue is composed of extracellular matrix and is located in the loading environment under low oxygen tension in knee joints. To form engineered constructs with mechanical properties compatible to native tissue, scaffolds should provide structural support, maintain cell phenotype and subsequently promote tissue development. The focus of this dissertation is on utilizing the physiological conditions found in joints to regulate biological behavior of cells. The first factor that was studied was the extracellular matrix. Two formats of silk fibroin-hydrogel and porous scaffolds - were examined for their potential as a supporting material for creating cartilage tissue constructs. The composite silk made from nano-fibers and hydrogel - a structure resembling the collagen network and proteoglycan in native cartilage - improved equilibrium and dynamic modulus of engineered tissue by 50% and 60%, respectively, in comparison to silk hydrogel without fibers. The second factor studied was the modulation of oxygen level, which is a major regulator during native cartilage development. Chondrogenic differentiation was induced in human embryonic stem cells under hypoxic conditions, in conjunction with biochemical cues from bovine chondrocytes. As a result, SOX9, a key transcription factor of cartilaginous lineage, was upregulated in the induced cells. Subsequent cultivation under normoxic conditions resulted in robust formation of cartilage tissue. Taken together, studies conducted in my thesis work address two major challenges in cartilage tissue engineering: i) providing cells with structural and mechanical properties similar to native ECM for generating in vitro cartilaginous tissue and ii) preconditioning cells with physiological environment for directing chondrogenic differentiation

Exploring the gelation mechanisms and cytocompatibility of gold (III)-mediated regenerated and thiolated silk fibroin hydrogels

Accelerating the gelation of silk fibroin (SF) solution from several days or weeks to minutes or few hours is critical for several applications (e.g., cell encapsulation, bio-ink for 3D printing, and injectable controlled release). In this study, the rapid gelation of SF induced by a gold salt (Au3+) as well as the cytocompatibility of Au3+-mediated SF hydrogels are reported. The gelation behaviors and mechanisms of regenerated SF and thiolated SF (tSF) were compared. Hydrogels can be obtained immediately after mixing or within three days depending on the types of silk proteins used and amount of Au3+. Au3+-mediated SF and tSF hydrogels showed different color appearances. The color of Au-SF hydrogels was purple-red, whereas the Au-tSF hydrogels maintained their initial solution color, indicating different gelation mechanisms. The reduction of Au3+ by amino groups and further reduction to Au by tyrosine present in SF, resulting in a dityrosine bonding and Au nanoparticles (NPs) production, are proposed as underlying mechanisms of Au-SF gel formation. Thiol groups of the tSF reduced Au3+ to Au+ and formed a disulfide bond, before a formation of Au+-S bonds. Protons generated during the reactions between Au3+ and SF or tSF led to a decrease of the local pH, which affected the chain aggregation of the SF, and induced the conformational transition of SF protein to beta sheet. The cytocompatibility of the Au-SF and tSF hydrogels was demonstrated by culturing with a L929 cell line, indicating that the developed hydrogels can be promising 3D matrices for different biomedical applications.This article has been prepared with the support of REMIX Project, funded by the European Union’s Horizon 2020 Research and Innovation programme under the Maria Sklodowska-Curie grant agreement N.778078. Chavee Laomeephol acknowledges the PhD grants supported by Chulalongkorn University for “The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship” and “The 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund)”. Authors also acknowledge the financial support from FCT (Portuguese Foundation for Science and Technology) for the project PTDC/CTM-BIO/4388/2014 - SPARTAN, the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000023 FROnTHERA) and the NORTE 2020 Structured Project, co funded by Norte2020

TRBP and eIF6 Homologue in Marsupenaeus japonicus Play Crucial Roles in Antiviral Response

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp

Involvement of Cyclin K Posttranscriptional Regulation in the Formation of Artemia Diapause Cysts

Background: Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood. Principal Finding: This study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb) in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2) in the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNA polymerase II (RNAP II). Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK) survival signaling pathway. Conclusions/Significance: Taken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role wa

On the routines of wild-type silk fibroin processing toward silk-inspired materials: a review

For years, silk fibroin of a domestic silkworm, Bombyx mori, has been recognized as a valuable material and extensively used. In the last decades, new application fields are emerging for this versatile material. Those final, specific applications of silk dictate the way it has been processed in industry and research. This review focuses on the description of various approaches for silk downstream processing in a laboratory scale, that fall within several categories. The detailed description of workflow possibilities from the naturally found material to a finally formulated product is presented. Considerable attention is given to (bio-) chemical approaches of silk fibroin transformation, particularly, to its enzyme-driven modifications. The focus of the current literature survey is exclusively on the methods applied in research and not industry

Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair

Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue-scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber-hydrogel composites (SF-silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber-hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mari. Excellent chondrocyte response was observed on SF-silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Transient hypoxia improves matrix properties in tissue engineered cartilage

Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days) and transient hypoxiareoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young's moduli of tissues grown under transient hypoxia (510.01 +/- 28.15kPa) and under normoxic conditions (417.60 +/- 68.46kPa) were significantly higher than those measured under hypoxic conditions (279.61 +/- 20.52kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs. (c) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 544553, 201

Vogt-Koyanagi-Harada Disease in Thailand

Purpose: To determine clinical characteristics and rate of ocular complications in patients with Vogt-Koyanagi-Harada disease (VKH). Methods: The authors performed a retrospective review of 48 consecutive patients (92 affected eyes) diagnosed with VKH. Demographic data, clinical manifestations, treatment modalities, and ocular complications were registered. Results: VKH constituted approximately 10% of all new encounters of uveitis patients. Ocular complications developed in 49/92 (53%) eyes. The most frequent complication was cataract (45%), followed by glaucoma (29%). The presence of ocular complications was not associated with stage of the disease at presentation (p = .654) or the treatment modalities (p = .261). Recurrent inflammation developed in 13% and was not associated with initial intravenous corticosteroids or use of immunosuppressive dr Conclusions: Cataract and glaucoma represented the common ocular complications of VKH. Recurrences developed solely in 13% of patients and were not associated with a specific treatment regimen but with an early withdrawal of systemic corticosteroids