Phospholipid‐induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration

Silk fibroin (SF) hydrogels can be obtained via self‐assembly, but this process takes several days or weeks, being unfeasible to produce cell carrier hydrogels. In this work, a phospholipid, namely, 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) sodium salt (DMPG), was used to induce and accelerate the gelation process of SF solutions. Due to the amphipathic nature and negative charge of DMPG, electrostatic and hydrophobic interactions between the phospholipids and SF chains will occur, inducing the structural transition of SF chains to the beta sheet and consequently a rapid gel formation is observed (less than 50 min). Moreover, the gelation time can be controlled by varying the lipid concentration. To assess the potential of the hydrogels as cell carriers, several mammalian cell lines, including L929, NIH/3T3, SaOS‐2, and CaSki, were encapsulated into the hydrogel. The silk‐based hydrogels supported the normal growth of fibroblasts, corroborating their cytocompatibility. Interestingly, an inhibition in the growth of cancer‐derived cell lines was observed. Therefore, DMPG‐induced SF hydrogels can be successfully used as a 3D platform for in situ cell encapsulation, opening promising opportunities in biomedical applications, such as in cell therapies and tissue regeneration.     

Effects of acid type on physical and biological properties of collagen scaffolds

Different acid types, hydrochloric acid and acetic acid, were used to prepare collagen solutions. The effects of acid types on physical and biological properties of collagen scaffolds were investigated. The collagen solution prepared with hydrochloric acid (C-HCl) was much more viscous than that prepared with acetic acid (C-Acetic). The conformation of collagen chains was expected to be different due to differences in ionic strength of collagen solutions. Morphology of the scaffolds analyzed by scanning electron microscopy (SEM) revealed that C-HCl scaffolds had larger pore sizes (around 100 microm) than C-Acetic scaffolds (around 50 microm). The more viscous C-HCl solution resulted in harder scaffolds shown by higher compressive modulus. However, the swelling ability of the C-Acetic scaffolds was higher in phosphate-buffered saline solution. The results from in vitro L929 cell culture showed that C-HCl scaffolds could promote cell initial attachment while the C-Acetic scaffolds enhanced cell proliferation. Therefore, the type of acid used as a solvent to form collagen solution affects the morphology, physical and biological properties of the resulting collagen scaffolds.     

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 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.     

Development of acellular dermis from porcine skin using periodic pressurized technique

In this work, a new method for producing acellular dermis (ADM), a natural scaffold used for dermal replacement, from porcine skin was developed. Fresh porcine skin from local slaughterhouse was dehaired by sodium sulphide following by epidermis removal using glycerol. After fat removal by chloroform/methanol (2/1 v/v) solvent, cellular components were removed using enzymatic treatment incorporated with a periodic pressurized technique. The effects of enzyme type (trypsin and dispase II) and periodic pressurized conditions on the efficiency of cell removal were investigated. When periodic pressure was applied, enzymatic treatment time could be shorten since the enzyme solution was able to penetrate into tight dermis. As a result, cells could be easily removed from porcine skin as noticed quantitatively by DNA assay and qualitatively by H&E staining. When enzyme refreshment was introduced into the decellularized process, the percentage of cell removal was further enhanced. This ensured that no inhibitions effect from the removed cells on enzyme-substrate interaction. Moreover, short-time enzymatic treatment with periodic pressurized technique could prevent the disruption of dermal structure, as observed by SEM. Dispase II can be used to remove cell better than trypsin in the periodic pressurized technique. However, in vivo study indicated that numerous fibroblast from the host tissue infiltrated into ADM prepared using both enzymes. Neo-collagen and neo-capillaries were produced in both implanted ADMs. The result elucidated that the use of periodic pressurized technique with enzymatic treatment has a high potential to be a new method to produce ADM for skin tissue engineering.     

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.     

Expansion of the mandibular arch in children during the mixed dentition period--a clinical study

A review of the dental literature revealed relatively few studies on the expansion of the mandibular dental arch. The present study attempted expansion of the mandibular arch using a Bihelix appliance. The subjects were 16 children, exhibiting crowding, age ranges from 7 to 11 years. The mandible was expanded 2.0 mm every 3 months. Significant expansion, not only of the individual tooth inter-arch dimensions but also of the overall arch length, was obtained during the period of incisor tooth replacement. The mode of expansion was classified as follows: Type I, those, which showed no effect on the apical base; Type II, those which showed no consistency of the measurement lines. In this study, 6 of 16 cases were classified as type I and 10 cases as type II. Expansion was continued over a period of 1.5 to 3 years. We concluded that considerable lateral expansion of the mandibular arch is possible using the Bihelix appliance. It is suggested that this might contribute greatly to non-extraction orthodontic treatment. Further studies are recommended.