Sorada Kanokpanont

The effect of sericin from various extraction methods on cell viability and collagen production.

Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

Monitoring of inflammatory mediators induced by silk sericin.

Silk proteins have been shown to be good candidates for biomedical materials. However, there have been some reports regarding immunological and allergic responses to silk sericin. Our objective was to investigate the inflammatory mediators induced by sericin both in vitro and in vivo. Mouse monocyte and alveolar macrophage cell lines were used for monitoring levels of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha generated after activation by sericin at concentrations of 0.2-1.0 mg/mL. The amounts of TNF-alpha and IL-1beta produced by both cell lines corresponded, in a dose-dependent manner, with the sericin concentration in the culture medium. The levels of TNF-alpha and IL-1beta generated after sericin activation by macrophage cells were higher than those generated by monocytes. However, these cytokine levels would not cascade to other inflammatory effects. Inflammatory mediators were also monitored from sericin-treated, cream base-treated and normal saline-soaked full-thickness rat excisions. Using wound size measurements and ELISA assays, sericin-treated wounds were shown to heal faster and had lower levels of inflammatory mediators, as compared with the cream base-treated and normal saline-soaked wounds. It can be concluded that sericin promotes the wound healing process without causing inflammation.

Influences of physical and chemical crosslinking techniques on electrospun type A and B gelatin fiber mats

This work has investigated the factors influencing the production of electrospun gelatin fibers including electrical potential and concentration of gelatin solution. Electrospun gelatin fibers were prepared from both type A and B gelatin solutions at the concentration of 2.5-60% w/v and 10-25 kV. Concentration of gelatin solution at 20-40% w/v was found to be the optimized range to produce the gelatin fibers with smooth surface throughout the fiber length. The electrical potential did not exhibit a dominant effect on the gelatin fibers obtained. Further study of the different crosslinking techniques for the gelatin fiber mats showed the various effects on the crosslinking degrees and fiber structure. Physical crosslinking such as dehydrothermal treatment, plasma treatment and their combination resulted in low crosslinking extent of gelatin fiber mats due to the crosslinking occurring only at the surface of the material. Combination of dehydrothermal and chemical crosslinking using 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride (EDC) or glutaraldehyde (GA) indicated higher crosslinking degree since both the surface and the bulk of the material were crosslinked. Spraying/immersion in EDC solution, a modified technique, resulted in swollen fibers while interconnected pores remained. Merged fibers were obtained from the crosslinking by GA vapor. We concluded that crosslinking is one of the key methods to control structure and degradation of the gelatin fiber mats. Various structures of gelatin fiber mats are expected to be useful for numerous applications.

The influence of molecular weight of chitosan on the physical and biological properties of collagen/chitosan scaffolds

Biopolymer blends between collagen and chitosan have the potential to produce cell scaffolds with biocompatible properties. However, the relationship between the molecular weight of chitosan and its effect on physical and biological properties of collagen/chitosan scaffolds has not been elucidated yet. Porous scaffolds were fabricated by freeze-drying the solution of collagen and chitosan, followed by cross-linking by dehydrothermal treatment. Various types of scaffolds were prepared using chitosan with various molecular weights and blending ratios. Fourier transform infrared spectroscopy proved that collagen and chitosan scaffolds at all blending ratios contained mainly electrostatic interactions at the molecular level. The compressive modulus decreased with increasing the concentration of chitosan. Equilibrium swelling ratios of approximately 6–8, determined in phosphate-buffered saline at physiological pH (7.4), were found in case of collagen-dominated scaffolds. The lysozyme biodegradation test demonstrated that the presence of chitosan, especially the high-molecular-weight species, could significantly prolong the biodegradation of collagen/chitosan scaffolds. In vitro culture of L929 mouse connective tissue fibroblast evidenced that low-molecular-weight chitosan was more effective to promote and accelerate cell proliferation, particularly for scaffolds containing 30 wt% chitosan. The results elucidated that the blends of collagen with low-molecular-weight chitosan have a high potential to be applied as new materials for skin-tissue engineering.

Formulation and characterization of silk sericin–PVA scaffold crosslinked with genipin

A porous-three-dimensional scaffold shows several advantages in terms of tissue engineering since it can provide a framework for cells to attach, proliferate and form an extracellular matrix. Sericin, a by-product from the silk industry, can form a three-dimensional scaffold with PVA after freeze-drying but has a fragile structure. Glycerin (as a plasticizer) and genipin (a crosslinking agent) are necessary to make a strong and stable matrix. Our objective was to investigate the properties of a three-dimensional silk sericin and PVA scaffold with and without glycerin and genipin at various concentrations. SEM showed that adding glycerin into scaffold gave better uniformity and porosity. Smaller pore sizes and better uniformity were found as the concentration of genipin in the scaffold increased. The results of FTIR indicated that glycerin retained a high moisture content and had a major effect at 3286 cm(-1), indicating the presence of water molecule in the matrix structure. Adding genipin into the scaffold resulted in a higher degree of crosslinking or fewer free ∈-amino groups, as shown by the decrease in the stretching (=C-H) peak and absorption peaks around 1370-1650 cm(-1), respectively. The sericin/PVA scaffold had a low water sorption capacity, but adding glycerin significantly increased this property. Genipin further enhanced the moisture absorption capacity of the scaffold and extended the time taken to reach equilibrium. After immersing the sericin/PVA scaffold into purified water, the scaffold completely dissolved within an hour, whereas the scaffolds containing glycerin or glycerin with 0.1% genipin swelled 8 and 11 times, respectively, compared with the initial stage after 6h of immersion. In terms of mechanical properties, the sericin/PVA/glycerin scaffold exhibited a similar compressive strength to the scaffold with a high genipin concentration, whereas a low concentration of genipin softened and reduced the compressive strength of the scaffold. A small amount of sericin was released from the scaffold and a higher concentration of genipin, resulting in less protein leaching compared to non-crosslinked sericin/PVA. The fraction of protein released from the sericin/PVA/glycerin scaffold was about 4%, with values of about 1 and 0.04% in the case of scaffolds with 0.01 and 0.1% genipin, respectively. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications.

An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing

In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silk fibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin and glutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroin fabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesive than the commercial wound dressing mesh. This confirmed by results of peel test on both the partial- and full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneous porous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularly for the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that the bi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed the greater extent of wound size reduction, epithelialization, and collagen formation. The superior properties of the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wound dressings were less adhesive and had improved biological functions to promote cell activities and wound healing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thickness wounds.

The Effect of Sericin with Variable Amino-Acid Content from Different Silk Strains on the Production of Collagen and Nitric Oxide

Although silk sericin (SS) enhances the growth and attachment of fibroblast cells, its toxicity remains questionable. We investigated the effect of SS extracted by heat with variable amino-acid content on in vitro collagen promotion and nitric oxide synthesis. After 24 h of incubation, SS, especially from the Chul 1/1 strain which has the most methionine and cysteine content, enhanced fibroblast growth. The molecular mass of heat-extracted SS from these three strains showed a slightly different range, but within 20–200 kDa, which were all identified as sericin. SS from all strains promoted type-I collagen production in a concentration-dependent manner, while SS from Chul 1/1 strain could induce the highest amount of collagen synthesis when compared to SS from other strains. Nitric oxide was found in the culture medium after activation by SS from the Chul 1/1 strain but reached a level that was not toxic to the cells. We conclude that SS is not toxic to fibroblast cells. Moreover, methionine and cysteine content in SS are important factors to promote cell growth and collagen synthesis.

Physico-chemical characteristics of methotrexate-entrapped oleic acid-containing deformable liposomes for in vitro transepidermal delivery targeting psoriasis treatment

This study aimed to investigate the physico-chemical characteristics and in vitro permeability of methotrexate (MTX)-entrapped deformable liposomes prepared from phosphatidylcholine (PC) and oleic acid (OA), comparing with those of MTX-entrapped conventional liposomes prepared from PC and cholesterol (CH). Two formulations of MTX-entrapped PC2:CH1 and PC9:CH1 liposomes and one formulation of MTX-entrapped PC2.5:OA1 liposomes were prepared. The size, size distribution, zeta potential, thermal properties, entrapment efficiency, stability, and in vitro permeability across a porcine skin of the MTX-entrapped liposomes were evaluated. All liposome formulations showed a narrow size distribution with the size range of 80-140 nm which is appropriate for the skin permeability. The percentage of MTX loading, entrapment efficiency and the stability of MTX-entrapped PC2:CH1 and PC9:CH1 liposomes were slightly higher than those of MTX-entrapped PC2.5:OA1 liposomes. However, the MTX-entrapped PC2.5:OA1 liposomes enhanced the skin permeability characterized by the higher concentration and flux of MTX diffused across or accumulated in the epidermis and dermis layers of porcine skin. The enhanced permeability of MTX-entrapped PC2.5:OA1 liposomes was explained by 2 mechanisms: (1) the deformable and elasticity characteristics of OA-containing liposomes and (2) a property as a skin penetration enhancer of OA. This suggested that the PC2.5:OA1 deformable liposome was one of promising candidates to enhance the permeability of MTX for the treatment of psoriasis.

Properties and antityrosinase activity of sericin from various extraction methods.

The present study investigated the chemical properties and antityrosinase activities of SS (silk sericin) extracted from different Thai silk strains via various extraction methods. Different silk strains contain distinct SS with various amino acid compositions, which are significantly influenced by the extraction method used. Urea extraction of SS was the only method that provided clearly distinguishable bands and had the most significant impact on SS conformation as illustrated by FTIR (Fourier‐transform infrared) spectra. The use of urea or either acidic or alkaline chemicals in the extraction process also influenced SS thermal behaviour. With regard to biological activity, SS extracted using urea exhibited the highest antityrosinase activity, whereas alkali‐degraded SS showed no inhibition of mushroom tyrosinase. Pigments, primarily flavonoids and carotenoids from silk cocoons, were also found to enhance tyrosinase inhibition of SS.

Preparation of Thai silk fibroin/gelatin electrospun fiber mats for controlled release applications

This work studied the effect of preparation conditions for electrospun fiber mats of Thai silk fibroin/type B gelatin (SF/GB) for controlled release applications. The increasing in applied voltage resulted in decreased average fiber size and narrow fiber size distribution. An increasing in silk fibroin content in blended solution resulted in larger size of the obtained electrospun fibers. Smooth fibers could be produced from SF/GB blended solution at weight blending ratios of 10/90, 20/80, 30/70, 40/60 and 50/50. The results on in vitro biodegradation test showed that SF/GB 10/90 electrospun fiber mat was rapidly degraded in collagenase solution due to direct biodegradation of gelatin by collagenase. From in vitro controlled release of two active agents (azo-casein and methylene blue) from SF/GB blended fiber mats, it was suggested that methylene blue could be adsorbed on the blended fiber mats, possibly due to attractive interaction of the positively charged molecules of methylene blue and negatively charged SF/GB fiber mats. In contrast, the same charge of blended fiber mats and azo-casein would result in the repulsive force, resulting in continuous diffusion of azo-casein from blended fiber mats within 72 h. The results indicated that SF/GB electrospun fiber mats had a high potential to be applied in controlled release applications.